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Multitarget, Selective Compound Design Yields Potent Inhibitors of a Kinetoplastid Pteridine Reductase 1

  • Ina Pöhner
    Ina Pöhner
    Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany
    Faculty of Biosciences, Heidelberg University, D-69120 Heidelberg, Germany
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    Orcidhttps://orcid.org/0000-0002-2801-8902
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  • Antonio Quotadamo
    Antonio Quotadamo
    Tydock Pharma srl, Strada Gherbella 294/B, 41126 Modena, Italy
    Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, 41121 Modena, Italy
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  • Joanna Panecka-Hofman
    Joanna Panecka-Hofman
    Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany
    Faculty of Physics, University of Warsaw, 02-093 Warsaw, Poland
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    Orcidhttps://orcid.org/0000-0003-4149-9090
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  • Rosaria Luciani
    Rosaria Luciani
    Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
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  • Matteo Santucci
    Matteo Santucci
    Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
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  • Pasquale Linciano
    Pasquale Linciano
    Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
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  • Giacomo Landi
    Giacomo Landi
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
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  • Flavio Di Pisa
    Flavio Di Pisa
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
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  • Lucia Dello Iacono
    Lucia Dello Iacono
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
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  • Cecilia Pozzi
    Cecilia Pozzi
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
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    Orcidhttps://orcid.org/0000-0003-2574-3911
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  • Stefano Mangani
    Stefano Mangani
    Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
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    Orcidhttps://orcid.org/0000-0003-4824-7478
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  • Sheraz Gul
    Sheraz Gul
    Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
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  • Gesa Witt
    Gesa Witt
    Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
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  • Bernhard Ellinger
    Bernhard Ellinger
    Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
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  • Maria Kuzikov
    Maria Kuzikov
    Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
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    Orcidhttps://orcid.org/0000-0001-8771-1865
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  • Nuno Santarem
    Nuno Santarem
    Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology, Universidade do Porto, 4200-135 Porto, Portugal
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  • Anabela Cordeiro-da-Silva
    Anabela Cordeiro-da-Silva
    Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology, Universidade do Porto, 4200-135 Porto, Portugal
    Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
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  • , 
  • Maria P. Costi*
    Maria P. Costi
    Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-0443-5402
  • , 
  • Alberto Venturelli*
    Alberto Venturelli
    Tydock Pharma srl, Strada Gherbella 294/B, 41126 Modena, Italy
    Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
    *Email: [email protected]
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  • , and 
  • Rebecca C. Wade*
    Rebecca C. Wade
    Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany
    Faculty of Biosciences, Heidelberg University, D-69120 Heidelberg, Germany
    Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, D-69120 Heidelberg, Germany
    Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, D-69120 Heidelberg, Germany
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-5951-8670
Cite this: J. Med. Chem. 2022, 65, 13, 9011–9033
Publication Date (Web):June 8, 2022
https://doi.org/10.1021/acs.jmedchem.2c00232

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure–activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.

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Introduction

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The World Health Organization has identified 17 neglected tropical diseases (NTDs) that pose a health burden to over 1.4 billion people. (1,2) Parasites of the trypanosomatid family are responsible for two potentially lethal insect-vector borne NTDs: human African trypanosomiasis (HAT, sleeping sickness), caused by Trypanosoma brucei, and leishmaniasis, caused by the intracellular parasite Leishmania spp. (3−7) Current therapeutics are limited by toxicity, poor efficacy, and parasite resistance, thus underlining the need for new chemotherapies. (8,9)
New antiparasitic agents can be identified by target-based drug design strategies. (10−12) The folate pathway enzyme dihydrofolate reductase (DHFR) is a known anticancer, antibacterial, and antimalarial target. (13−16) It provides reduced folates, which are crucial to biological processes like DNA, protein, and amino acid synthesis or one-carbon transfer. (14,17,18) In trypanosomatids, DHFR inhibition, for example by methotrexate (MTX, 1a), is ineffective due to a metabolic bypass via the biopterin-reducing pteridine reductase 1 (PTR1, Figure 1): when DHFR is inhibited, PTR1 is overexpressed and sustains sufficient reduced folate levels to ensure parasite survival. Thus, when targeting the folate pathway in Leishmania, both DHFR and PTR1 need to be considered. (19−21) In T. brucei, RNA interference studies have suggested PTR1 to be a potential antiparasitic target in its own right. (22,23) Nonetheless, even nanomolar PTR1 inhibitors have so far shown limited antiparasitic activity in vitro, (24,25) suggesting that targeting the T. brucei folate pathway may also benefit from the consideration of both PTR1 and DHFR.

Figure 1

Figure 1. Overview of pterin activation in the trypanosomatidic folate pathway when DHFR is inhibited and PTR1 provides a metabolic bypass. Under normal conditions (indicated by dashed lines), the DHFR domain of the bifunctional DHFR-TS reduces biological folates to tetrahydrofolate (THF). Serine hydroxymethyl transferase (SHMT) converts THF to 5,10-methylene THF, which has a central role in amino acid synthesis, protein biosynthesis, and one-carbon transfer. It is also required by the TS domain of DHFR-TS to convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), which is necessary for DNA synthesis. PTR1 catalyzes the reduction of unconjugated pterins, like biopterin, and takes over folate reduction when DHFR is inhibited (continuous lines), thus acting as a metabolic bypass and an important additional target for shutting down the trypanosomatidic folate pathway. Both proteins are shown in cartoon representation (DHFR domain of DHFR-TS: purple, PTR1 monomer of the functional tetramer: light pink) with the NADPH/NADP+ cofactor in a stick representation with black carbons and the folate substrate in yellow spheres. In PTR1, an arginine residue from a neighboring subunit that points into the active site is shown in a magenta stick representation.

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Screening a set of folate-related compounds against parasitic folate pathway targets previously led to the identification of compounds 1b (methyl-1-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzoyl)piperidine-4-carboxylate) and 1c (methyl-1-(4-(((2,4-diaminopteridin-6-yl)methyl)amino)benzoyl)piperidine-4-carboxylate) as submicromolar inhibitors of Leishmania major PTR1 (LmPTR1) with Ki values of 0.04 and 0.10 μM, respectively. (26)1c was additionally a micromolar inhibitor of L. major DHFR (LmDHFR) with a weak selectivity for the parasite enzyme over the human DHFR (hDHFR) (Ki of 4 vs 10 μM). In contrast to the parasite DHFR, which is covalently coupled with thymidylate synthase (TS) in a bifunctional DHFR-TS, the hDHFR off-target is monofunctional and shares only about 30% sequence identity with parasite DHFR domains, indicating potential for further optimization of selectivity. (27−29)
The current study focuses on optimizing pteridine-based compounds for their inhibition of T. brucei PTR1 (TbPTR1) and TbDHFR, in addition to the corresponding Leishmania targets, while ensuring selectivity against the off-target hDHFR. The enzymatic evaluation of reference pteridines reported earlier, (26,30) our comparative study of trypanosomatid folate pathway proteins, (31) and computational docking studies were first employed for the design of novel pteridine derivatives. Three new crystal structures of complexes of pteridines with TbPTR1 and a complex with LmPTR1 were determined and confirmed the predicted bound orientation of the novel pteridines. A systematic analysis of correlations between computed physicochemical molecular descriptors and observed antiparasitic effects was then performed and allowed us to prioritize promising compounds for synthesis. In total, we identified 26 new pteridine-based multitarget inhibitors showing improved target inhibitory profiles for PTR1 and DHFR of both L. major and T. brucei. Among these inhibitors, we report the first, to the best of our knowledge, apparent picomolar inhibitors of TbPTR1 and several new low nanomolar inhibitors of LmPTR1, which mostly also show selective micromolar to submicromolar inhibition of the parasite DHFR variants. In vitro evaluations of the designed multitarget inhibitors against bloodstream forms of T. brucei brucei revealed low micromolar to submicromolar EC50 values for three of these pteridines. Taken together, we here report a successful application of a systematic multitarget design approach to yield selective pteridine-based antiparasitic compounds affecting multiple trypanosomatid enzymes.

Results and Discussion

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Reference Compounds Inhibit both PTR1 and DHFR

To systematically assess multitarget inhibition, we measured the inhibition of TbPTR1, TbDHFR, LmPTR1, LmDHFR, and the off-targets hDHFR and hTS by the folate-related anticancer agent methotrexate (MTX, 1a) and seven further pteridine-based reference compounds (1b–1h, Figure 2 and Table S1, SI). (26,30,32) Although 1b–1h were primarily designed as LmPTR1 inhibitors, we found all to be more potent against TbPTR1 than LmPTR1 with 1b being the strongest inhibitor of TbPTR1 with an IC50 of 50 nM against TbPTR1 and 1 μM against LmPTR1 (Figure 2A). Notably, these compounds exhibited micromolar to submicromolar inhibition of LmDHFR and TbDHFR (IC50LmDHFR 0.3–1.4 μM; TbDHFR 0.3–20.05 μM). While MTX (1a) was more potent against the parasite DHFRs, it was not selective (selectivity index SI: TbDHFR/hDHFR = 3 and LmDHFR/hDHFR = 1, Figure 2A). For compounds 1b–1h, higher SI values were observed, ranging up to about 165 for 1b for both TbDHFR and LmDHFR (Figure 2A).

Figure 2

Figure 2. Inhibitory activities, selectivities, and structures of reference pteridines. (A) Heatmaps show activities given by IC50 values (top) and selectivity indices (SI) (bottom) for the targets and the off-target hDHFR. All values, as well as data for hTS, are given in Table S1. NI: no inhibition; NA: not applicable. (B) Previously published compounds shown were used as reference compounds: 1a is methotrexate; 1b, 1c, and 1h are 6b, 6a, and 6c from Cavazzuti et al.; (26) and 1d–1g correspond to 5d, 5b, 6a, and 5a from Corona et al. (30)

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Substrate-like and Methotrexate-Inhibitor-like Binding Modes of the Reference Compounds

Despite the hydrogen-bonding network stabilizing the pteridine ring in the PTR1 active site, for the PTR1 complexes with MTX/1a derivatives, there are two alternative binding modes. Previously determined crystal structures show that compounds 1b and 1c share a substrate-like pterin orientation in the complex with LmPTR1. (26) In the same crystal structure, compound 1b also adopts a second, so-called inhibitor-like (or MTX-like) orientation, with the bicyclic ring system flipped by 180° and rotated by 30° (Figures 3A,B and S1). (26) Dual binding modes have also been observed in crystallographic complexes of TbPTR1 with small pteridine-based inhibitors. (32)

Figure 3

Figure 3. Orientations of reference pteridine compound 1b in crystal structures of LmPTR1 and TbPTR1. (A,B) Compound 1b (cyan carbons) in complex with LmPTR1 (PDB-ID 2qhx) has a substrate-like (A) and an inhibitor-like or MTX-like (B) binding mode. 1b is shown with (A) folate (yellow carbons) superimposed from a TbPTR1 structure (PDB-ID 3bmc) and with (B) MTX (1a, yellow carbons) superimposed from an LmPTR1 structure (PDB-ID 1e7w). The pteridine nitrogens are labeled according to the ring nomenclature. (C) Binding site in the crystal structure determined in this work (PDB-ID 6rx5) of TbPTR1 (gray cartoon, His267′ from the neighboring subunit in lavender) in complex with NADPH/NADP+ and compound 1b, which has the MTX-like binding mode. Interacting residues (in A, B: only Phe113) and the NADPH/NADP+ cofactor are shown in sticks (carbons colored according to protein and black, respectively). In (C), water molecules are shown as red spheres, and the inhibitor is surrounded by the omit map (green wire) contoured at the 2.5 σ level. Hydrogen bonds are represented by brown dashed lines.

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We here determined the crystal structure of the ternary complex of TbPTR1 with NADPH/NADP+ and the reference compound 1b (PDB-ID 6rx5, resolution 1.42 Å, experimental details: Tables S2 and S3). It shows that the diaminopteridinyl moiety of 1b adopts only the MTX-like orientation (Figure 3C), resembling its MTX-like binding mode in LmPTR1 (Figures 3B and S2). Consistently, docking studies indicated that all reference pteridines adopt MTX-like binding modes in the different targets and the off-target hDHFR (Table S4 and Figure S5). Therefore, we concluded that the MTX-like binding mode is likely the dominant one, and we focused on the analysis of this binding mode in the subsequent compound design.

Comparative Target/Off-Target Mapping and Docking Studies Support Design Focused on Selective Multitarget Inhibition

To develop enhanced selective inhibitors of the parasite targets, we employed a multitarget-based design approach to improve inhibition of TbPTR1, TbDHFR, LmPTR1, and LmDHFR while retaining low hDHFR off-target inhibition. The next generation of pteridine-like compounds was created by dissecting the part of 1b attached to the pteridine core into three modules. These were N10, the substitution to the N10 position; PABA, the para-amino benzoic acid (PABA) moiety; and Tail, the cyclic glutamate tail (Figure 4). We separately modified each of these modules to obtain three new series of compounds. The modifications of each module were based on binding mode predictions from docking in the different targets and the off-target hDHFR and our previously published optimization guidelines for MTX-like scaffolds. (31) The key concepts adopted in the compound design are summarized in Figure 4.

Figure 4

Figure 4. Structural features of PTR1 and DHFR considered in the multitarget design of selective compounds illustrated for reference compound 1b. (A) Selected residues within 5 Å of the three modules─N10, PABA and Tail─modified in the design procedure. Residues were selected for the complexes of 1b with TbPTR1 (pale gray), TbDHFR (dark gray), LmPTR1 (pale pink), and LmDHFR (dark pink). Residues are colored according to their properties: basic: blue, polar: green, and nonpolar: yellow. The ligand interaction plot is based on Panecka-Hofman et al. (31) and provides an overview of residues with similar properties that surround the ligand modules in the different targets (showing only those applied for the design; for full maps, see Figures S3 and S4). In some positions, the amino acid type of the off-target hDHFR is different from parasite DHFR. Differing hDHFR residues are labeled in the top right corner of the corresponding parasite DHFR residue. These positions highlight suitable substitution points to improve selectivity. (B) Surface representations of complexes of 1b with TbPTR1 (left, PDB-ID 6rx5) and TbDHFR (right, MTX-like top-ranked docking pose in PDB-ID 3rg9). The compound tail moiety is fully solvent-exposed in PTR1, whereas it is well-enclosed in DHFR. (C) Surface representations of complexes of 1b with TbPTR1 (left, PDB-ID 6rx5) and LmPTR1 (right, PDB-ID 2qhx, state A). The ligand is more enclosed in the narrow pocket entrance of TbPTR1, while the LmPTR1 pocket has an elongated, widened funnel that can accommodate larger compound tails. In (B,C), 1b is shown in sticks with cyan carbons.

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Rationale for N10 Modifications

The binding pockets of the different target proteins were found to share a number of aliphatic residues in the proximity of the N10 substituent of a bound ligand, e.g., Leu209 of TbPTR1; Ile47 and Leu90 of TbDHFR; Leu226 and Leu229 of LmPTR1; Ile20 and Val62 of LmDHFR (Figure 4A). (31) Bulkier nonpolar groups in comparison to the methyl of 1b, like the ethyl and propargyl substituents of 2a and 2b, allow for interactions with those hydrophobic moieties. Docking studies suggested that even substituents of the size of benzyl, as in 2c, can be accommodated in the PTR1 and DHFR pockets (Figure 5A,B). Furthermore, such bulky substituents may improve selectivity for the on-targets: The hDHFR pocket has a lower volume compared to the parasite DHFR pockets (pocket volume TbDHFR 353 Å3, LmDHFR 384 Å3, and hDHFR 347 Å3).

Figure 5

Figure 5. Views of the binding sites showing docked poses of selected pteridine-based inhibitors in the target proteins: TbPTR1 (pale gray) (A,E), TbDHFR (dark gray) (B,F), LmPTR1 (pale pink) (C), and LmDHFR (dark pink) (D). (A) Induced fit (IF) MTX-like docking pose for compound 2c (cyan carbons) in TbPTR1 in the presence of a conserved water molecule (ball-and-stick representation): Trp221 moves (indicated by a brown arrow) to make room for the phenyl of 2c. (B–F) Rigid-body docking poses of 2c in TbDHFR (B), 3c (lime carbons) in LmPTR1 and LmDHFR (C,D), and 4e (purple carbons) in TbPTR1 and TbDHFR (E,F); see text for discussion. Docked poses are shown for N1-deprotonated compounds, but similar orientations were observed for the N1-protonated forms (see Figure S6). For PTR1, all docking poses shown were obtained in the presence of conserved structural water molecules. Generally, similar poses were observed for docking without water. In all panels, proteins are shown in cartoon representation with the important interacting residues (compare Figure 4A) and the NADPH/NADP+ cofactor shown in sticks (carbons colored according to protein and black, respectively). Residues His267′ and Arg287′ from the neighboring subunit are shown in lavender and magenta in TbPTR1 and LmPTR1, respectively. Hydrogen bonds are represented by brown dashed lines. Further IF docking poses are shown in Figures S7 and S8.

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Furthermore, as previously demonstrated, (31) hDHFR favors hydrogen bond donors in the proximity of N10 and the PABA ring system, whereas the parasite DHFRs allow for favorable interactions with hydrogen bond acceptors. To improve off-target selectivity, we thus replaced N10 by sulfur and the PABA benzene ring by pyridine in 2d.
Although Corona et al. (30) found improved selectivity for PTR1 over hDHFR by hydrophilic N10 substitutions, our data for reference compound 1d with a hydroxyethyl substituent did not support this observation (Figure 2A). Docking simulations indicated that interactions with a highly conserved structural water might induce an unfavorable conformation of the substituent’s aliphatic chain (Figure S5A, SI). To relax the geometry while allowing interactions between the substituent and water, we elongated the aliphatic linkage to a hydroxypropyl in 2e.
Thus, in total, the N10 series consists of five novel pteridines (2a–e, Figure 6) modified to improve interactions with PTR1 and parasite DHFR and to exploit the differences in pocket sizes and residues between the parasitic targets and the hDHFR off-target.

Figure 6

Figure 6. Overview of the modifications in the N10, PABA, and Tail modules explored in the designed compound series with respect to the reference compound 1b. Synthesized members of each designed series are shown in the framed boxes along with the key objectives addressed with the respective modifications. See text for details.

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Rationale for PABA Modifications

As a first modification to the PABA moiety, in 3a, we replaced the PABA phenyl group with benzyl (Figure 6). The additional hydrophobic spacer can interact with hydrophobic target residues while resulting in a shifted position of the hydrophilic linker amide. The positioning of hydrophobic and hydrophilic residues surrounding the PABA moiety and the amide linker in the human off-target is different from that of the parasite targets PTR1 and DHFR, which can be exploited to improve selectivity. For the same reason, the amide linker position was also shifted in 3b by substituting the PABA moiety with meta-aminobenzoic acid.
A second key feature of the targets vs off-targets that was used to inform the design of the PABA series relates to the compound tail: Tail regions are solvent-exposed in PTR1 and thus have poorly defined interactions (Figure 4B). In contrast, in DHFR, the tail region is enclosed, and strong interactions occur with the hDHFR off-target. (31) We therefore shortened the tail region to achieve full enclosure in the PTR1 binding pocket by replacing PABA by naphthalene (3c) or benzene moieties (nonsubstituted, 3d; or substituted with −CF3, 3e). Docking results showed that the smaller tail fully resides in the PTR1 binding pocket (Figure 5C) and is stabilized by surrounding hydrophobic residues, not only in PTR1 but also in parasite DHFR (Figure 5C,D). Rigid-body docking studies suggested that the bulky naphthalene of 3c may be particularly beneficial in LmPTR1, since this target has a more elongated, open pocket compared to TbPTR1 (Figure 4C). The PABA moiety modifications are therefore suitable for modulating the compound interaction profile in a species-specific manner.
In summary, the PABA series contains five new pteridine derivatives (3a–3e, Figure 6) designed to improve selectivity by exploiting the different surroundings of bound PABA moieties in hDHFR in comparison to the parasite target proteins.

Rationale for Tail Modifications

The surrounding of the compound tail features several hydrophobic residues, particularly in the two T. brucei targets (Figure 4A). Directional interactions with the tail moiety may have limited benefit for the binding affinity in PTR1, since the flexibility of the solvent-exposed tail likely has an entropic contribution. Hydrophobic interactions are geometrically less restrained than, for instance, hydrogen bonds. Therefore, anticipating less pronounced entropic penalties on binding in our designed derivatives, we replaced the methyl ester in the tail of 1b by the more flexible ethyl and propyl in 4a and 4b, respectively. Two aspects may result in selectivity benefits from this approach: The tail region is enclosed by more hydrophobic moieties in parasite DHFR than in the hDHFR off-target (Figure 4A), and residues surrounding the tail have previously been demonstrated to show differing conformational variability in the crystal structures when comparing parasitic targets with the off-target. (31)
To combine exploitation of the differing patterns of hydrophobic residues in the tail environments of targets and off-targets with improved enclosure in PTR1 (Figure 4A,B), we further modified the tail to an unsubstituted piperidine (4c) or replaced piperidine with an unsubstituted benzene (4d). Compound 4e, with benzene attached via a flexible ethyl linkage to an MTX-like amide, can benefit from nonpolar and aromatic residues surrounding the tail in PTR1 and parasite DHFR and, according to docking predictions, readily adapt to their differing placements in the on-targets; see Figure 5E,F. The docking studies additionally suggest that the flexible aromatic tail can form cation−π interactions with positively charged residues in the entrance of the DHFR pocket (e.g., Arg59 of TbDHFR, Figures 4A and 5F). Additional hydrophobic residues in the target pocket entrance regions, like Pro99 of TbPTR1 (Figure 4A), can be targeted with an altered geometry in combination with methoxylations: 4f and 4g combine a one-carbon spacer between N10 and PABA and amide-linked methoxylated tail portions. In addition, an etheryl linkage to a nonsubstituted (4h), methoxylated (4i), or trimethoxylated (4j) benzyl group was explored. Compounds 4f–4j were collectively designed to interact with the different hydrophobic, aromatic, and positively charged surrounding residues found around the tail region in the various targets (Figure 4A).
Taken together, the tail series comprises 10 new pteridines (4a–4j, Figure 6) with modified tails to target residue patterns distinguishing on- and off-targets and the distinct surroundings of the tail in PTR1 vs DHFR.

Synthesis of Pteridine Derivatives with High Yield

A total of 26 new 2,4-diaminopteridine derivatives and the reference compounds 1b and 1c were (re)synthesized as reported in Schemes 1–8. We applied our methodology for an improved reaction yield of the chemical pteroid step to provide a key intermediate for most of the designed compounds. (33) Displacement of the chloride of 6-(chloromethyl)pteridine-2,4-diamine hydrochloride (29, Scheme 1) by the appropriate substituted anilines and aliphatic amino derivatives was carried out in N,N-dimethylacetamide (DMA) at 60 °C microwave (MW) to provide 1b,c, 2a–e, 3a–c, 4a–j, and 5a–f in high yields of 70–90% with reduced reaction time (Schemes 2–7). (33)

Scheme 1

Scheme 1. Synthesis of Derivatives of Compound 29a
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aReagents and conditions: (i) SOCl2, reflux, 12 h, 70% yield; (ii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 60°C, 20′–30′ MW.

Scheme 2

Scheme 2. Synthesis of Compounds 1b,c, 2a–c, 2e, 4a–c, and 5c, and Intermediates 32–35, 37, 38, and 40–49a
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aReagents and conditions: compounds 30, 31, 36, and 39 were purchased from Sigma; (i) acetonitrile or 3-hydroxypropanenitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24–36 h (32, 33); (ii) alkyl halide (propargyl bromide, (bromomethyl)benzene) (0.5 equiv), K2CO3 (2 equiv), DMF dry, rt, 24 h (34, 35); (iii) SOCl2 (4 equiv), propanol (for 37), EtOH (for 38), reflux, 7–12 h (89 and 96% yield); (iv) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (40–49); (v) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (1b,c, 2a–c, 2e, 4a–c, 5c).

Scheme 3

Scheme 3. Synthesis of Compounds 3a and 5a,ba
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aReagents and conditions: (i) 3-hydroxypropanenitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24 h (51); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (52–54); (iii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (3a, 5a,b).

Scheme 4

Scheme 4. Synthesis of Compounds 4f,ga
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aReagents and conditions: (i) di-tert-butyl pyrocarbonate (1.05 equiv), dioxane/H2O/1 N NaOH 1/1/1 V/V/V, rt, 6 h (55); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (56 and 57); (iii) TFA, DCM, rt (58 and 59); (iv) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (4f,g).

Scheme 5

Scheme 5. Synthesis of Compounds 4h–j and 5e,fa
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aReagents and conditions: (i) K2CO3 (3 equiv), DMF, reflux, 16–18 h (63–65); (ii) NH2OH·HCl (1.2 equiv), EtOH, rt, >1 h followed by Zn dust (2.5 equiv) in 12 M HCl (4 equiv), rt, 15′ (66–68); (iii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (4h–j, 5e,f); (iv) methylamine (for 69) or benzylamine (for 70), EtOH dry, 60°C, 3 h, then NaBH4 (1.5 equiv), rt, 2 h.

Scheme 6

Scheme 6. Synthesis of Compounds 3b and 2da
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aReagents and conditions: (i) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (71 and 72); (ii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (3b, 2d).

Scheme 7

Scheme 7. Synthesis of Compounds 3c, 4d,e, and 5da
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aReagents and conditions: (i) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 30′ MW (3c, 4d,e, and 5d); (ii) acetonitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24–36 h (74); (iii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (75).

Scheme 8

Scheme 8. Synthesis of Compounds 3d,ea
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aReagents and conditions: (i) KMnO4, acetone/0.5 M phosphate buffer at pH 7 (1:1 V/V); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight.

The PABA amine functionalization was achieved by selective alkylation of primary amines to secondary amines using nitriles as alkylating reagents with Pd/C for intermediates 32 and 33. (34,35) Conventional alkylation of the latter with propargyl bromide or (bromomethyl)benzene resulted in derivatives 34 and 35, respectively (Scheme 2).
The reductive alkylation of amines using nitriles was also used to obtain 51 and 74 in Schemes 3 and 7. The isonipecotic acid derivatization was achieved via Fischer esterification using the reagent solvents propanol (37) and EtOH (38), respectively; methyl isonipecotate (36) and piperidine (39) were purchased from Sigma (Scheme 2).
The intermediate acid derivatives 30–35 and d and e were condensed to amides through a coupling reaction with the respective amines 36–39 and g using EDC·HCl in dimethylformamide (DMF) as the coupling agent to provide the intermediate products 40–49, 71, 72, and 75, which were then made to react with 29 to obtain the final compounds (1b,c, 2a–d, 3b, 4a–c, 4e, 5c; Schemes 2, 6, 7).
Using the same method, we synthesized the elongated compounds 3a, 4f–g, and 5a,b, characterized by a carbon spacer in the PABA moiety (Schemes 3 and 4).
To obtain 4f,g (Scheme 4), an additional protection step reaction to guide selective amide functionalization was necessary. The selectivity was achieved via Boc protection in the first step of the reaction of b to obtain 55, which was then coupled with the respective aliphatic amine to give 56 and 57. The target amines were finally obtained by a deprotection step carried out in 30–40% trifluoroacetic acid/dichloromethane (TFA/DCM) in quantitative yield.
The phenoxyphenyl–methanamine derivative intermediates (Scheme 5) were synthesized starting from 4-fluorobenzaldehyde and the respective phenol derivates 60–62 by an SNAr reaction. Subsequently, the primary amines 66–68, (36) or functionalized amines 69 and 70 (obtained via a one-pot reductive step), were reacted with 29 to obtain 4h–j and 5e,f.
Compounds 3c, 4d, and 5d, with a higher steric hindrance, were obtained with a slightly increased reaction time in a good yield. Finally, to obtain 3d,e, it was necessary to first perform an oxidation reaction. Treatment of Pt–OH in acetone/0.5 M phosphate buffer at pH 7 (1:1 v/v) with KMnO4 gave the oxidized analogue 76, which was subsequently coupled with the selected aliphatic amine to obtain the desired amides (Scheme 8).

Crystal Structures for the PTR1 Targets Confirm the Predicted Interactions and That the Pteridine Derivatives Adopt a Methotrexate-Inhibitor-Like Orientation

The structures of TbPTR1 with two new pteridines, 2a and 2e, and that of LmPTR1 with 2e, were determined to 1.20, 1.11, and 2.10 Å resolution, respectively (see Tables S2 and S3). The structures contain functional enzyme tetramers in the crystallographic asymmetric unit with a similar structure to those previously determined. (37,38) In all complexes, the compounds adopt MTX-like binding modes (Figure 7A,B).

Figure 7

Figure 7. Views of the binding sites of crystal structures of complexes of pteridine-based inhibitors in TbPTR1 and LmPTR1 determined in this work, which confirm the predicted MTX-like binding modes. (A) 2a (green carbons) in TbPTR1 (gray cartoon, His267′ from the neighboring subunit in lavender) and (B) 2e (yellow carbons) in LmPTR1 (pink cartoon, Arg287′ from the neighboring subunit in magenta). Water molecules are shown as red spheres, and the inhibitors are surrounded by the omit map (green wire) contoured at the 2.5 σ level. Interacting residues and the NADPH/NADP+ cofactor are shown in sticks (carbons colored according to protein and black, respectively). Hydrogen bonds are represented by brown dashed lines.

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In line with the docking predictions, the overall structure of the TbPTR1 complexes resembles the complex with 1b (compare Figure 7A with 3C). In agreement with the design objective, the N-ethyl moiety of 2a was found to form van der Waals interactions with Val206 and Trp221 on the hydrophobic side of the pocket (Figure 7A). The bulkier N-propylhydroxyl moiety of 2e forms direct and water-mediated hydrogen bonds with Asp161 and receives an intramolecular hydrogen bond from the amine in position 4 on the pteridine system (Figure S2C). The structure of LmPTR1 in complex with 2e (Figure 7B) closely resembles that observed in TbPTR1, except for the terminal piperidine moiety (Figure S2C,D). The latter moiety is highly flexible─a possible orientation is reported in the crystal structure, but further orientations cannot be excluded.

Designed Pteridine Derivatives Have Improved Target and Off-Target Enzyme Inhibitory Activities

The measured inhibitory activities of compounds 2a–e, 3a–e, and 4a–j against the targets TbPTR1, TbDHFR, LmPTR1, and LmDHFR and the off-targets hDHFR and hTS are given in Figure 8 and Table S1. All inhibitory activities are reported as IC50 values, which are commonly used to characterize and rank compounds when screening for enzyme inhibition in drug discovery projects. (39) Overall, the inhibitory activities against the PTR1 targets for the designed compounds are improved, as are PTR1 vs off-target selectivities. Indeed, for a small number of compounds (2c, 2d, 4c, 4d, 4e, 5c, and 5d), the IC50 values for inhibition were determined to be either 1 nM or less than 0.1 nM against TbPTR1. As the TbPTR1 assay makes use of low nanomolar concentrations of enzyme, for these very potent compounds, the tight binding limit was approached, and therefore, accurate values of the IC50 values could not be determined. (40) Representative dose–response curves are shown in the Supporting Information, showing that only part of the response range could be measured for these compounds for which the IC50 value could also be rather sensitive to any possible errors in dilution or determination of inhibitor or enzyme active site concentration.

Figure 8

Figure 8. Inhibitory activities (IC50 values, left) and selectivities (selectivity indices (SI), right) of compounds of the designed N10-, PABA-, and Tail-modified series and selected reference compounds against the targets TbPTR1, LmPTR1, TbDHFR, and LmDHFR and the off-target hDHFR. All values, as well as data for hTS, are reported in Table S1. Greener boxes show higher inhibition and selectivity. $ indicates that a precise activity value could not be determined as the tight binding limit was approached.

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N10 Modifications Yield Improved PTR1 Inhibitors with Similar Selectivity Trends for Parasite DHFRs

The N10-modified compounds (2a–e; Figure 6) are improved PTR1 inhibitors in comparison to 1b, except for 2a (1b IC50TbPTR1 50 nM, LmPTR1 1 μM; N10 series IC50TbPTR1 < 0.1–90 nM; LmPTR1 0.02–13.3 μM; Figure 8). 2c is the best in the series with IC50 < 0.1 nM against TbPTR1 and an IC50 of 20 nM against LmPTR1.
All compounds are roughly similar to 1b in parasite DHFR inhibition (1b IC50TbDHFR and LmDHFR 0.3 μM; N10 series IC50TbDHFR 0.4–2.4 μM, LmDHFR 0.5–9.4 μM), and selectivities over hDHFR range from 7- to 66-fold for TbDHFR and 9- to 110-fold for LmDHFR, which are somewhat lower than for 1b (SI TbDHFR/hDHFR = 164 and LmDHFR/hDHFR = 167). Thus, mainly PTR1 inhibition benefits from the selected N10 modifications.

PABA Modifications Lead to Strong Variations in the Target Inhibition Profile

The modifications of the PABA moiety in the PABA series (compounds 3a–e; Figure 6) distinctly affect the inhibitory activities against the targets. Smaller compounds with well-enclosed binding poses show varying improvements in inhibitory activity for different PTR1 variants: 3c, in contrast to most of the studied pteridines, is equipotent toward LmPTR1 and TbPTR1 (IC50 10 nM). This notable improvement of LmPTR1 activity is in line with its predicted good steric fit to the LmPTR1 binding pocket shape (compare Figures 4C and 5C). Full enclosure and stabilizing interactions with hydrophobic residues lining the pocket entrance likewise probably contribute to an around 10-fold higher potency of 3d and 3e against TbPTR1 than the most similar reference compound 1c (lacking an N10 substitution) (IC503d, 3e: 10 nM; 1c: 110 nM).
Whereas 3d and 3e do not show inhibitory activity against the parasite DHFR targets, 3c shows similar activity against LmDHFR to 1c (IC50 1.5 and 0.6 μM, respectively) and displays higher activities against both TbDHFR (IC501c: 20.5 μM; 3c: 0.5 μM) and hDHFR (IC501c: 51 μM; 3c: 4 μM). A one-carbon spacer to shift the position of the PABA carbonyl in 3a with respect to 1c improves inhibition of TbDHFR (IC50 0.6 μM) while not significantly affecting inhibition of LmDHFR and hDHFR. Thus, 3a is more selective against TbDHFR than the reference 1c (SI: 56 vs 2).
Taken together, alterations to the PABA moiety, due to its central location in the compound scaffold, different pocket sizes, and surrounding residue patterns in targets (Figure 4A) display highly variable effects on the activity profiles.

Alterations in Tail Geometry Boost PTR1 Inhibition but Can Reduce DHFR Inhibition

In the Tail-modified series (compounds 4a–j; Figure 6), hydrophobic and aromatic residues lining the pocket entrance region of PTR1 were exploited by either tail elongation or shortening. The interactions of these residues with the flexible aromatic tail of 4e (see Figure 5E) likely contribute to the boost of the IC50 against TbPTR1 to the subnanomolar range and to 30 nM against LmPTR1; these are 1000-fold and 57-fold improvements, respectively, in PTR1-inhibitory potencies compared to reference compound 1c. The shortened tails of 4c (unsubstituted piperidine) and 4d (benzene) are stabilized by the same residues and likely benefit from a better enclosure in the PTR1 pocket. Both compounds show improved TbPTR1 and LmPTR1 inhibition compared to 1b (IC50TbPTR1 4c: 10 nM, 4d: 1 nM vs 1b: 50 nM; LmPTR1 4c: 0.1 μM, 4d: 0.03 μM vs 1b: 1.0 μM).
However, shortening of the tail diminishes the inhibition of parasite DHFR, whereas it either does not affect or increases inhibition of the off-target hDHFR. Revisiting the docking predictions provides a possible explanation for this: The piperidine/benzene groups in the tails of 4c and 4d can form more extended hydrophobic interactions with Phe31 of hDHFR than with the corresponding methionine in the parasite DHFR variants (Figure 9). In the parasite protein, moreover, Asn64 in the pocket entrance of hDHFR is replaced by phenylalanine, which, upon interaction with the compound tail, becomes solvent-exposed.

Figure 9

Figure 9. Docking poses for compound 4c from the Tail series (magenta carbons) in (A,B) TbDHFR and (C) hDHFR, showing differences in exposure and interactions of the PABA and Tail moieties in the two DHFRs. (A) TbDHFR pocket accommodates 4c with its tail enclosed by surrounding residues. hDHFR has a similar shape. TbDHFR is shown in a gray surface representation. (B,C) Views of the binding sites of TbDHFR and hDHFR, which are shown in cartoon representation in gray and green, respectively. Important interacting residues and the NADPH/NADP+ cofactor (black carbons) are shown as sticks. Hydrogen bonds are indicated by brown dotted lines. While the orientations of 4c are rather similar in both DHFR variants, the tail moiety is more solvent-exposed in TbDHFR: the PABA benzene and piperidine of 4c compete for interactions with Phe94 of TbDHFR, which thereby becomes exposed to the solvent. In hDHFR, the corresponding exposed residue is the polar Asn64, and the tail of 4c can interact with Phe31 deeper in the pocket, rendering the mode of binding more favorable in hDHFR. The results are presented for N1-deprotonated compounds, but similar observations were made with N1-protonated compounds (Figure S6).

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Pocket size and interaction pattern differences between LmDHFR and other DHFR variants, as also discussed for the PABA series, also affect the Tail-modified compounds: for instance, 4d is more active against both TbDHFR and hDHFR than 1c (IC50TbDHFR 1 vs 21 μM, hDHFR 6 vs 51 μM), while both compounds show similar activity for LmDHFR.

Summary of the Compound Activity Profiles for the N10, PABA, and Tail-Modified Series

Taken together, most of the new pteridine derivatives display 1–2-fold greater inhibition of TbPTR1 than LmPTR1 and are more or equally active against PTR1 than the reference compound 1b. The nanomolar to subnanomolar PTR1 inhibitors show improved selectivity for PTR1 over the off-target hDHFR by up to about 3 orders of magnitude (2c, 4e: TbPTR1 IC50 < 0.1 nM; SI > 400 000) (Figure 8). The IC50 against hDHFR is typically greater than 100 μM, whereas inhibitory activities against TbDHFR and LmDHFR are higher. For parasite DHFR, the compounds with the best inhibitory activities have similar IC50 values to 1b (e.g., LmDHFR IC504a: 0.13 μM, 4b: 0.15 μM, and 1b: 0.26 μM). Thus, the newly designed compounds show improved target inhibitory profiles, particularly for the PTR1 targets, and overall good selectivity for the parasitic proteins.

Inhibitory Activity against T. brucei Is Related to the Hydrophobicity of the Compounds

Following the assessment of the improvement on the target inhibition level, we next determined the antiparasitic effect on T. brucei brucei Lister 427 bloodstream forms and L. infantum intramacrophage amastigotes (Figure 10A and Table S7). The LmPTR1 and LmDHFR proteins are highly similar to the corresponding L. infantum proteins (91 and 96% sequence identity, respectively), but in spite of the improved effect on both target proteins, the designed pteridines are mostly inactive against L. infantum. In contrast, the compounds show activity against T. brucei.

Figure 10

Figure 10. Antiparasitic activity expressed as percentage of inhibition against T. brucei brucei for reference compounds and members of the N10-, PABA-, and Tail-modified series (A) and the selected representatives of the merged in silico library (B). The average of at least three independent determinations is shown with the standard deviation. The inactive compounds in the Tail-modified series, 4f, 4h, and 4j were omitted. Activities can be found in Table S7.

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The multiple correlation coefficient between the TbPTR1 and TbDHFR IC50 values and the T. brucei bloodstream form inhibition is R = 0.35 (eq 3, SI), indicating that the levels of target enzyme inhibition are, for the current compounds, only weakly correlated with the exhibited antiparasitic effect when assuming a linear correlation. PTR1 inhibition alone shows a Pearson correlation R = 0.34 with T. brucei inhibition, whereas R is only 0.24 for DHFR inhibition, possibly because all studied compounds are much stronger inhibitors of PTR1 than DHFR. The low correlation for DHFR inhibition might also arise because of the competition from the high folate concentration in the medium in the parasite assays.
Another reason for the low correlations between parasite and target protein inhibition could be transport issues. For example, the charged compound tail and possible polyglutamylation of the parent MTX (1a) have previously been suggested to influence compound transport. (41,42) All the newly designed pteridines lack the glutamate tail, which may affect their in vivo activities, but there might be other structural or physicochemical features that render them more or less active against parasites despite similar target inhibition. For example, for T. brucei, we noticed that while 2c and 4e have similar effects on the targets TbPTR1 (0.1 nM) and TbDHFR (approximately 1 μM), they differ notably in their inhibitory effect on the parasite bloodstream forms (75 vs 40%).
Therefore, we investigated the correlations of physicochemical properties and ADMET predictors with the measured effect on T. brucei; see Table 1. Our aim was to identify which properties were indicative of a better antiparasitic effect, possibly related to better uptake. Overall, only weak correlations of the individual properties with T. brucei inhibition were observed (Pearson R: 0.47–0.55 and −0.41 – −0.54; computed as defined in the SI). The strongest correlation was found for the predicted skin permeability, QPlogKp, as a descriptor linked to lipophilicity, (R: 0.55). The logPo/w and the binding to human serum albumin had slightly weaker correlations with the antiparasitic effect (R: 0.49 and 0.47, respectively). For these properties, an increase in the value corresponds with higher anti-T. brucei activity. In contrast, some properties showed anticorrelation, for instance, the aqueous solubility and the cohesive index (44) (R: −0.54 and −0.41, respectively). Taken together, the data indicate an improved antiparasitic effect with increased lipophilicity of the studied compounds.
Table 1. Descriptors with Significant Correlations with the Observed Inhibitory Effect on T. brucei for the Reference Compounds and Pteridines of the N10-, PABA-, and Tail-Modified Series Calculated with QikProp (43)a
predicted propertyQPlogKpQPlogPo/wQPlogKhsacohesive indexCIQPlogS
R0.550.490.47–0.41–0.54
R20.300.240.220.170.29
P-value0.0030.010.010.040.004
resampling recovery rate (%)10096965696
optimization direction↑↑↑↓↓
covered range–6.62 – −3.60–1.02–2.92–0.85–0.350.02–0.04–6.71 – −3.19
recommended range–8.00 – −1.00–2.00–6.50–1.50–1.500.00–0.05–6.50–0.50
a

QPlogKp: Predicted skin permeability, log Kp; QPlogPo/w: Predicted octanol/water partition coefficient. QPlogKhsa: Prediction of binding to human serum albumin. Cohesive index: Index of cohesive interaction in solids, (number of hydrogen bond acceptors × number of hydrogen bond donors × 0.5/surface area); (44) CIQPlogS: Conformation-independent predicted aqueous solubility, log S with S in mol dm–3 being the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid. R (Pearson correlation) and R2 were calculated using the percentage of inhibition of the T. brucei brucei Lister 427 bloodstream form at a 10 μM compound concentration as defined in the SI. Only descriptors with at least a Pearson correlation/anticorrelation of 0.40/–0.40 and two-tailed P-values lower than the chosen significance level α of 0.05 are reported. The covered range lists property values obtained for the studied compounds, while the recommended range lists values the properties take for typical drug-like molecules. The resampling recovery rate indicates in how many cases (expressed as percentage) the same property was identified when leaving a single compound out of the data set. The optimization direction indicates whether higher or lower values would putatively lead to improved anti-parasitic effects.

Combined Modifications Yield Pteridines with Both Improved Target Inhibition and Improved Antiparasitic Activity

To explore further derivatives of the studied pteridines, we next designed a merged compound library as follows. The pteridine core scaffold was retained, and the studied compounds were decomposed into fragments of their N10, PABA, and Tail regions and recombined in silico in all possible combinations to yield 2014 derivatives (see SI for details). These derivatives were evaluated in docking studies against targets and off-targets and additionally prioritized by the physicochemical marker properties that showed correlations with the anti-T. brucei effect (Figure S9). Of the remaining 600 candidates, 6 were selected by expert opinion as representative compounds for synthesis and experimental evaluation (5a–5f, Figure 11).

Figure 11

Figure 11. Inhibitory activities, selectivities, and structures of the merged series of six pteridine derivatives. (A) Activity heatmap in the top panel shows IC50 values for the targets TbPTR1, LmPTR1, TbDHFR, and LmDHFR and the off-target hDHFR. All values, as well as data for hTS, are reported in Table S1. $ indicates that a precise activity value could not be determined as the tight binding limit was approached. In the bottom panel, selectivity indices are reported. (B) Structures of the selected and synthesized pteridines in the merged series.

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Two compounds, 5a and 5b, were chosen for their favorable interaction patterns and scores predicted by docking simulations. 5a combines the N10 hydroxypropyl fragment of 2e, benzyl in place of the PABA phenyl of 3a, the tail amide of reference compound 1g, and, for 5b, in addition, the tail pyrrolidine of ref 1h, which replaces the tail piperidine. The activities and predicted interactions in all parasite targets are most similar to 2e, suggesting the key importance of the hydroxy-propyl substituent to N10 for the target inhibition. Notably, while 5a is poorly selective for TbDHFR (2-fold) and modestly selective for LmDHFR (31-fold), 5b is inactive against hDHFR, resulting in SI values of 169 and 113 for TbDHFR and LmDHFR, respectively. Moreover, 5b has SI values over hDHFR of about 25 000 for TbPTR1 and 588 for LmPTR1. However, in contrast to most compounds, 5b displays a weak inhibition of hTS (IC50 29 μM, Table S1).
Four additional compounds (5c–5f, Figure 11) were prioritized based on the physicochemical marker properties. Compound 5c combines fragments of ethyl modification to N10 of 2a and the tail ethyl ester of 4a. Due mainly to the tail ester, this modification improves the inhibition for both TbPTR1 (IC50 1 nM) and LmPTR1 (IC50 0.1 μM). The activity against TbDHFR is similar to that of the N10-modified parent 2a, whereas LmDHFR and hDHFR inhibition are again influenced by the tail modification (IC50LmDHFR 5c: 0.2 μM, 4a: 0.1 μM; hDHFR 5c: 13 μM, 4a: 12 μM). Compound 5d merges the ethyl N10 fragment of 2a with the unsubstituted benzene of 4d. In TbPTR1, this boosts the nanomolar IC50 of 4d to the subnanomolar range, while the activity toward LmPTR1 remains similar to 4d. This profile can be related to the N10 ethyl, which seems disfavored in LmPTR1 as judged by the modest inhibition of the parent 2a (IC50 13.3 μM).
Compounds 5e and 5f combine the ethylphenyl(4-methoxyphenyl) ether scaffold of 4i with the benzyl and methyl N10 modifications from 2c or 1b, respectively. Both compounds are nanomolar inhibitors of both PTR1 variants. The parent compounds, 2c and 1b, inhibit the parasite DHFR variants at micromolar to submicromolar levels, while 4i is inactive against all variants of DHFR. The combination with a favorable N10 substitution is able to restore medium micromolar anti-DHFR activity for the altered scaffold of parent 4i in the parasite enzymes in 5e and 5f. Thus, combined N10 and tail modifications allowed for the species-specific optimization of the target inhibition profile.
Compounds 5d–5f show an improved percentage of T. brucei inhibition at 10 μM, in line with their selection for synthesis being motivated by altered marker properties (Figure 10B). For these compounds, EC50 values were determined; see Table 2. Indeed, the more lipophilic compounds were found to have low micromolar EC50s against T. brucei brucei, with 5d being the best (EC50 0.66 ± 0.48 μM), and they have SIs of 3–38 based on their cytotoxicity on THP-1-derived macrophages.
Table 2. Properties with a Significant Correlation with the Observed Inhibitory Effect on T. brucei for Compounds in the Merged Series Calculated with QikProp (43)a
compoundQPlogKpQPlogPo/wQPlogKhsacohesive indexCIQPlogS%inhibition of T. brucei at 10 μM ± SDEC50T. brucei [μM] ± SDCC50 [μM]selectivity index
5a–6.74–1.160.050.04–4.5330 ± 8N.D.N.D.N.D.
5b–6.48–1.320.430.04–6.3523 ± 4N.D.N.D.N.D.
5c–5.182.020.040.03–5.3257 ± 10N.D.N.D.N.D.
5d-3.912.190.070.02–5.4378 ± 30.66 ± 0.4825 < CC50 < 5038
5e–4.603.36–1.230.02–3.20100 ± 04.53 ± 0.4212.5 < CC50 < 253
5f–5.162.09–1.140.02–3.44100 ± 01.30 ± 0.0512.5 < CC50 < 2510
pentamidineN.D.N.D.N.D.N.D.N.D.N.D.0.0019 ± 0.0005105263
a

The properties are defined as in Table 1. Values shown in bold face are within 90% of the previously determined top value or exceeded the previously obtained range for the reference compounds and compounds in the N10-, PABA-, and Tail-modified series; see Table 1. The activity against the T. brucei brucei Lister 427 bloodstream form at a 10 μM compound concentration (%inhibition) is given. For the most promising compounds, 5d–5f, in addition, measured EC50 values, CC50 interval estimations, and selectivity indices are reported and compared to pentamidine, a reference compound with activity against T. brucei. EC50 represents the arithmetic average of at least two independent measurements done in triplicate. CC50 estimation was done by at least three independent cytotoxicity assessments on THP-1-derived macrophages by a colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, as previously reported. (45) The selectivity index is determined as the CC50 or lower CC50 interval estimation divided by EC50. N.D.: Not determined.

Bulky Compounds with Hydrophobic Substituents Often Display Liabilities

Potential liabilities were assessed by determining the inhibition of the hERG potassium channel as well as five isoforms of CYP450 (1A2, 2C9, 2C19, 2D6, and 3A4), cytotoxicity against A459 cells (human lung adenocarcinoma epithelial cell line), and mitochondrial toxiticity against 786-O cells (renal carcinoma cell line) for all compounds at a concentration of 10 μM. The results are shown in Figure 12. Further, the compounds were assessed for and passed a check for being pan-assay interference compounds (PAINS).

Figure 12

Figure 12. Heatmap representation of the liability assessment results for all the compounds studied. Inhibition of hERG as well as five CYP isoforms (1A2, 2C9, 2C19, 2D6, and 3A4), mitochondrial toxicity (MITO), and growth inhibition of A549 cells were determined at 10 μM. The data are represented as percentages on a color scale from white (desired) to orange (undesired) with values reported in the map. For the inhibitory activities against hERG, CYP isoforms, and mitochondrial toxicity, white = 0% and orange = 100% inhibition/toxicity, while for A549 cell growth inhibition, white = 100% and orange = 0% growth. The values are reported in Tables S8 and S9.

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The reference compounds and the N10 series mostly exhibit a safe profile. In contrast, aromatic modifications to the compound tail region, for instance in 3d, 4j, and 5f (PABA, Tail, and Merged series, respectively) were associated with notable hERG liabilities. Increasing the hydrophobicity of the compounds further led to liabilities against some CYP isoforms, in particular, 2C9 and 2C19. The shortened tails of 3c and 3d resulted in a strong effect on CYP isoform 2D6. Finally, several of the bulky, more hydrophobic compounds resulted in a cytostatic or cytotoxic effect on A549 cells. Overall, the liability assessment suggests that increasing hydrophobicity is associated with greater compound liabilities.
In line with these observations, two of the best inhibitors of T. brucei bloodstream forms, 5e and 5f, show 54 and 81% hERG inhibition, respectively. 5e, and in many cases 5f, affects various CYP isoforms. Finally, 5f is cytostatic with A549 cell growth reduced to 15%, and 5d shows cytotoxicity, effectively completely inhibiting cell growth. Thus, the most active inhibitors of T. brucei bloodstream forms were found to suffer from liabilities associated with their greater hydrophobicity and would require careful optimization of their cellular specificity.

Conclusions

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We applied a multitarget-based approach to the development of novel therapies for HAT and leishmaniasis, in which we focused on pteridine-based inhibitors of L. major and T. brucei PTR1 and DHFR, and successfully designed the first known apparent picomolar inhibitors of TbPTR1. While LmPTR1/LmDHFR inhibition was previously explored for this compound class, (26) we here demonstrated the potential of pteridine-based inhibitors against TbPTR1 and TbDHFR. We solved a crystal structure of the reference compound 1b bound to TbPTR1 to confirm the overlap in observed binding modes between LmPTR1 and TbPTR1 and the preference of the methotrexate inhibitor-like bound orientation in TbPTR1. Guided by our detailed comparative study of on- and off-targets in the parasitic and human folate pathway, (31) crystal structures of reference compounds, and enzymatic evaluation of published reference pteridines, (26,30) we designed 26 new pteridine derivatives that mostly have improved activity and selectivity. For their synthesis, we made use of an advanced MW-assisted protocol to improve the reaction yield of the pteroid step with reduced reaction time compared to previous synthetic procedures. (33) Further determination of the crystal structures of complexes and computational docking enabled us to obtain a complete characterization of the binding modes of the pteridines to their molecular targets and supported the derivation of a SAR. The compounds were also tested against the human off-targets hDHFR and hTS. While they were sometimes only modestly selective for the parasitic DHFR variant, many showed 1000-fold and higher selectivities for PTR1 over the off-targets and thus, the novel PTR1 inhibitors can overall be considered selective for the parasite proteins.
While many compounds exhibited excellent inhibitory activity at the target level, they were often only modest inhibitors of T. brucei brucei bloodstream forms and inactive toward L. infantum intracellular amastigotes in vitro. We found that increased lipophilicity correlated with improved inhibitory effects on T. brucei. We were able to prioritize compounds for synthesis from a designed combinatorial in silico library by using predicted ADMET-related properties, which suggested a likely improvement of the trypanocidal effect. In this way, we identified three improved compounds, 5d, 5e and 5f, with low micromolar inhibition of T. brucei brucei (EC50 0.66–4.53 μM).
The modulated on-target/off-target activities and selectivities of the above compounds showed that specific combinations of the N10 and tail modifications allow a fine-tuning of the target inhibition profile for enzymes of specific parasite species. Furthermore, the strategy employed here of combining property prediction correlation with multitarget-based compound design was found to be a useful approach to discovering antiparasitic agents, even when the antiparasitic data are available only as a percentage of inhibition determined at a single compound concentration. We also note that the presence of high folate concentrations in the HMI-9 medium used in the parasite assays may have resulted in underestimated antiparasitic activity due to competition between folic acid and folate-analogue inhibitors of DHFR. However, our main aim in this work was to identify potent PTR1 inhibitors with antiparasitic activity that are capable of targeting multiple enzymes. In future work, these compounds can be progressed to more in-depth mechanistic studies in various parasite and mice models. Further, integration of transport-related considerations in the design, (31) or using, for instance, structurally related scaffolds reported in the literature, which show inhibition of the Leishmania parasite, and a similar property-based correlation concept to that presented here, may help to overcome the current limitations of the pteridine-based compounds as inhibitors of intracellular parasites. Our data show that, overall, optimization for increased lipophilicity leads to more potent pteridine-based T. brucei inhibitors. However, increased lipophilicity can also introduce compound liabilities, e.g. for hERG and CYPs. Strategies to avoid these, for instance by making use of a similar property-based optimization strategy, should thus be incorporated in future design efforts.

Experimental Procedures

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General Synthesis Information

Reagent grade chemicals and solvents were purchased from commercial suppliers and used without further purification. Reactions were monitored by TLC on silica gel plates (Kieselgel 60, F254, Merck) and visualized using UV light, cerium ammonium sulfate, or alkaline KMnO4 aqueous solution. Solvents are abbreviated as follows: tetrahydrofuran (THF), ethyl ether (Et2O), dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), ethyl acetate (EtOAc), dichloromethane (DCM), dimethylformamide (DMF), methanol (MeOH), and acetonitrile (ACN). The structures of the isolated compounds were confirmed by NMR spectroscopy and mass spectrometry. NMR spectra were recorded on Bruker 400 and 600 spectrometers with 1H at 400.134–600 MHz and 13C at 100.62–151 MHz and are given in the Supporting Information. The purity of all synthesized compounds was determined by elemental analyses performed on a PerkinElmer 240C instrument and liquid chromatography–mass spectrometry (LC–MS) on UHPLC–HRMS (Agilent 6500 QTOF mass spectrometer) under electrospray ionization mode, with 4800 V of ion voltage at Centro Interdipartimentale Grandi Strumenti, CIGS UniMoRe). The purity of the reported compounds is >95%. Exact monoisotopic masses are reported in the Supporting information along with the melting point intervals of all compounds, which were measured on a Stuart SMP3 instrument.

General Synthetic Procedure A: Reductive Alkylation of Amines Using Nitriles (32, 33, 51, 74)

After two vacuum/H2 cycles to remove air from the reaction tube, the stirred mixture of the amine (1.0 equiv), Pd\C catalyst (10 wt % of the amine), the respective RCN (5.0 equiv), and NH4OAc (1.0 equiv) in MeOH (5.0 mL) was hydrogenated under ambient pressure (balloon) at room temperature (rt) for the appropriate time (24–36 h). The reaction mixture was filtrated using Celite cake before the filtrate was concentrated under reduced pressure. The residue was partitioned between Et2O (10 mL) and water (10 mL). The aqueous phase was extracted thrice with Et2O (10 mL), and the combined organic phases were washed with brine (10 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to yield the amines without further purification.

General Synthetic Procedure B: Amide Coupling Reaction for the Synthesis of 27, 28, 40–49, 52–54, 56, 57, 71, 72, 75

Carboxylic acid compounds (1 equiv), EDC·HCl (1.1 equiv), and HOBt (0.1 equiv) were added to a dried round-bottomed flask and dissolved in DMF dry under N2. The reaction mixture was cooled down to 0 °C and stirred for 30 min before it was added to the respective amine (1 equiv) with/without TEA (2–3 equiv). After mixing overnight at rt, the mixture was washed 1× with saturated NaHCO3, 1× with H2O, and 1× with brine. The washed organic mixture was then dried with Na2SO4, concentrated in vacuo, and purified using column chromatography (SiO2, eluent: Cy/EtOAc or DCM/MeOH or DCM/EtOAc/MeOH) to give the desired amide.

General Synthetic Procedure C: MW Alkylation 1b,c, 2a–e, 3a–c, 4a–j, 5a–f

To a suspension of amine intermediates (1 equiv) in DMA (3 mL) in a microwave Biotage vial, 29 (1.2 equiv), K2CO3 (3 equiv), and KI (0.1 equiv) were added. The vial was sealed and heated by microwave irradiation in a Biotage Initiator+ microwave at 60 °C for 20 min (30′ for compounds 3c, 4d, and 5d), before cooling to rt and diluting with water (20 mL). The precipitate was then collected by filtration and dried before the final compound was purified by fractional crystallization from methanol, DCM, and Et2O.

General Synthetic Procedure D: SNAr for the Preparation of 4-Substituted Benzaldehyde (63–65)

A mixture of substituted phenol 58-60 (1 equiv), 4-fluorobenzaldehyde (1 equiv), and K2CO3 (3 equiv) in DMF (10 mL) was refluxed for 16–18 h under nitrogen. After cooling, the solution was concentrated in vacuo to give a crude residue, which was purified by crystallization in 1 M NaHCO3. The obtained crystal was washed with H2O to obtain the desired benzaldehyde derivatives.

General Synthetic Procedure E: Preparation of Primary Amines from 4-Substituted Benzaldehyde (66–68)

A solution of carbonyl (aldehyde) compounds 66–68 (1 equiv) and hydroxylammonium chloride (1.2 equiv) in ethanol (30 mL) was stirred for 1 h at rt. Subsequently, 12 M hydrochloric acid (4 equiv) and zinc dust (2.5 equiv) were slowly added to the solution and left to stir at rt for 15 min. To the resulting slurry, a solution of ammonia (30%, 14 mL) and sodium hydroxide (6 M, 30 mL) was added dropwise, and the mixture was stirred at rt for another 15 min. Then, the resulting solution was extracted with DCM, dried over anhydrous Na2SO4, and filtered. The solvent was removed under vacuum to give the amines without further purification.

Protein Expression and Purification

Recombinant TbPTR1, LmPTR1, TbDHFR-TS, LmDHFR-TS, hDHFR, and hTS were expressed and purified according to previously reported procedures. (26,45,46)

Crystallization of TbPTR1 and LmPTR1

Well-ordered monoclinic crystals of histidine-tagged TbPTR1 were obtained by the vapor diffusion hanging drop technique at rt. (47) Drops were prepared by mixing equal volumes of protein and precipitant solution (2–2.5 M sodium acetate and 0.1 M sodium citrate pH 5) according to a previously described procedure. (45) The TbPTR1–cofactor–inhibitor ternary complexes were obtained by the soaking technique. The compounds, solubilized in DMSO, were diluted in the cryoprotectant solution (30% vol/vol glycerol added to the precipitant solution) to a final concentration of 2–4 mM (keeping the DMSO concentration below 10% vol/vol). Crystals were then transferred in the resulting soaking/cryoprotectant solution and flash frozen in liquid nitrogen after 8–24 h of exposure.
Crystals of LmPTR1 were prepared as described elsewhere. (38) The LmPTR1–cofactor–2e ternary complex was obtained by the soaking technique via the addition of 2 mM compound (solubilized in DMSO, without exceeding the 10% drop volume) directly into the crystallization drop. After 5 h, crystals were transferred to the cryoprotectant solution and flash frozen in liquid nitrogen.

Data Collection, Structure Solution, and Refinement

X-ray crystallographic data were collected using synchrotron radiation at the Diamond Light Source (DLS, Didcot, United Kingdom) beamlines I04-1 and I03 equipped with a Dectris Pilatus 6M-F and a Pilatus3 6 M detector, respectively. Reflections were integrated using MOSFLM and scaled with Scala (CCP4 suite). (48−52) Data collection and processing statistics are reported in Table S2. The crystals of TbPTR1 and LmPTR1 belonged to the primitive monoclinic space group P21 and the primitive orthorhombic space group P212121, respectively. Both had a functional enzyme tetramer in the asymmetric unit. The structures were solved by molecular replacement using MOLREP and either a TbPTR1 (PDB-ID 5jdc) or a LmPTR1 tetramer (PDB-ID 5l4n) as the searching model (all nonprotein atoms were excluded). (38,45,53) Models were refined using REFMAC5 (CCP4 suite). (54) Visual inspection and manual rebuilding of missing atoms was performed using Coot. (55,56) Water molecules were added with the automated standard procedures implemented in the software ARP/wARP and checked with Coot. (57) In the higher-resolution complexes of TbPTR1 with compounds 2a and 2e, all atoms were refined anisotropically in the final refinement cycles, and hydrogen atoms were added in the calculated positions. The occupancies of exogenous ligands were individually adjusted to values resulting in atomic displacement parameters comparable to those of surrounding protein atoms in fully occupied sites. The final models were checked with Coot and Procheck. (58) Statistics for data refinement are reported in Table S3. Figures were generated using CCP4mg. (59) Coordinates and structure factors were deposited in the Protein Data Bank under the PDB-IDs 6rx5 (TbPTR1-NADPH/NADP+-1b), 6rx0 (TbPTR1-NADPH/NADP+-2a), 6rx6 (TbPTR1-NADPH/NADP+-2e), and 6rxc (LmPTR1-NADPH/NADP+-2e).

TbPTR1, TbDHFR, LmPTR1, LmDHFR, hDHFR, and hTS Target/Off-Target Enzyme Assays

In vitro assays for TbPTR1 and LmPTR1 were based on the coupled assay reported by Shanks et al. (60) The assay nonenzymatically links the reduction of cytochrome c (Cc) with the reduction of dihydrobiopterin to tetrahydrobiopterin, catalyzed by PTR1. The formation of reduced Cc (Fe2+) results in a signal increase in the photometric readout at 550 nm wavelength. TbPTR1 and LmPTR1 assays were performed in a buffer containing 20 nM sodium citrate (pH 6.0) in a well-plate-based format as previously reported. (45)LmDHFR, TbDHFR, hDHFR, and hTS activities were assessed spectrophotometrically according to published procedures. (61,62) Each inhibitory compound was assayed at 11 different concentrations in triplicate, and IC50 values were calculated as described in the SI.

Computational Preparation of Pteridine Compounds and Protein Receptors and SiteMap Calculation of DHFR Pocket Volumes

The 3D structures of the reference and designed compounds were generated from SMILES strings and optimized with the OPLS_2005 force field using LigPrep of Maestro (Schrödinger, LLC) as described previously, except that tautomers were created for the pH range 5.0–8.0, and both N1-deprotonated and N1-protonated tautomers were considered for every compound. (45,63−66) In addition, all different substituents to the N10 position, PABA modifications, and compound tail alterations present in compounds 1b–4j were combined in all possible permutations in silico in a “merged” series and prepared similarly.
All receptors were prepared in the presence of MTX (from the following PDB-IDs for TbPTR1: 2c7v, LmPTR1: 1e7w, TbDHFR and LmDHFR: 3cl9 and hDHFR: 1u72) to improve the interactions of binding site residues and the conserved water molecules with the pteridine core. Receptor preparation was following published procedures with minor modifications. (45,63,64,67−69) For the LmPTR1 (PDB-ID 1e92) and TbPTR1 (PDB-ID 2x9g) receptors, an energy minimization with a harmonic restraint of 25 kcal mol–1 Å–2 on heavy atoms and no restraint on hydrogens was performed until the heavy atom RMSD relative to the previous minimization step was less than 0.30 Å. (70) For the TbDHFR receptor, PDB-ID 3rg9 was used; for LmDHFR, our previously published homology model based on a TcDHFR-TS template (PDB-ID 3inv) was chosen. (31) For off-target docking, we used the hDHFR structure 1u72. For PTR1, we also considered the previously described set of conserved water molecules identified by a WatCH clustering approach. (45,67) Further, using WatCH, we identified conserved water sites in hDHFR as described in the SI. (67) Except for the parasite DHFR variants, where the identification of a conserved water set was not possible, all receptors were prepared both with the identified set of conserved structural waters and without explicit water molecules. Grid preparation was done as described before for LmPTR1 and TbPTR1 (45) with the following grid centers and rotatable groups: (i) LmPTR1: center Phe113, rotatable OH in Ser111, Thr184, Tyr191, Tyr194, Thr195, Tyr283, and NADP+ ribose; (ii) TbPTR1: center Phe97, rotatable OH/SH in Ser95, Cys168, Tyr174, and NADP+ ribose; (iii) LmDHFR: center Phe31, rotatable OH/SH in Thr35, Thr36, Ser61, Cys130, Tyr137, Thr155, and NADP+ ribose; (iv) TbDHFR: center Phe58, rotatable OH in Thr46, Thr62, Thr86, Ser89, Ser98, Tyr166, Thr184, and NADP+ ribose; and (v) hDHFR: center Phe34, rotatable OH in Thr38, Thr39, Ser59, Tyr121, and NADP+ ribose.
The volumes of the binding pockets of TbDHFR (PDB-ID 3rg9), hDHFR (1u72), and the LmDHFR homology model were computed with Schrödinger SiteMap (63,71,72) as described in the SI.

Computational Docking Studies

Docking studies were performed using a rigid receptor in Glide standard precision (SP) and extra precision (XP) modes and employing the Induced Fit (IF) protocol to allow for refinement of binding site residues. (63,73−79) For rigid receptor docking, van der Waals radii scaling of ligand atoms and settings for sampling, addition of Epik state penalties to the docking score, rewarding of intramolecular hydrogen bonds, and enhancement of the planarity of conjugated π-groups were chosen as described previously, (45) but a total of 50 poses per ligand were subjected to postdocking energy minimization. For the in silico library, we used SP docking with a constraint on all heavy atoms of the pteridine core to match the orientation of MTX in the corresponding protein receptor with a tolerance of 1 Å.
In addition, since some compounds showed major variation in substituent size when compared to the starting scaffold and explicit water molecules are treated as frozen in the standard SP/XP docking, additional studies allowing protein side chain and water reorganization in response to ligand binding were performed using the standard protocol for the IF workflow implemented in Maestro. The planarity of conjugated π-groups was enhanced, and a Prime refinement was performed for residue side chains within 5 Å of ligand atoms. XP redocking was done as previously described, yielding up to 20 receptor–ligand complexes per compound. (45)
The validation of the docking protocol is presented in the SI.

Computational Property Prediction, Pan-assay Interference Compounds (PAINS), and Correlation Analysis with Antiparasitic Data

Physicochemical descriptors and parameters related to ADMET were computed for all prepared compounds using QikProp (Schrödinger). (43) Pearson correlations (R), R2 values, and two-tailed P-values for each property with the measured percentage of inhibition of T. brucei at the 10 μM compound concentration were computed using SciPy and Python scripts written for the purpose. Only properties with a P-value equal to or below the statistical significance level α = 0.05 were considered further. To ensure robustness, a resampling analysis was performed by leaving every compound out once before recomputing the correlations. Properties with R > 0.40 or < −0.40 and a P-value ≤ α in >50% of the resampling correlation analyses were considered to be the most robust markers for the optimization for antiparasitic effect. These properties were employed to prioritize compounds for synthesis as part of the Merged series; for details, see the SI and Figure S9.
In addition, a multivariate correlation coefficient between parasite target protein inhibition and antiparasitic activity was computed; for details, see the SI.
Finally, all synthesized compounds were checked for PAINS filters A, B, and C, undesirable substructure moieties, covalent inhibition, and compliance with the rule-of-five with the FAF-Drugs4 Web server (https://fafdrugs4.rpbs.univ-paris-diderot.fr) by inputing SMILES strings for the compounds. (80)

In Vitro Biological Evaluation against T. brucei and L. infantum Intramacrophage Amastigotes

The efficacy against T. brucei brucei Lister 427 bloodstream forms was evaluated in a modified resazurin-based assay as previously described. (81) Cells were grown at 37 °C and 5% CO2 in a complete HMI-9 medium supplemented with 10% fetal calf serum (FCS) and 100 UI/mL of penicillin/streptomycin. The HMI-9 medium was selected due to its high folic acid content (9 μM), resulting in efficient parasite growth and enabling process standardization. (82) The same medium was used in our previous experiments and allowed maintenance of similar relative folate metabolism levels between models studied in high- and low-throughput systems, namely, between Trypanosoma cells and mouse models, and between mouse models and human plasma. (83,84) Cultures were then diluted to a cell density of 2 × 106 /mL. For the assay, compounds were prepared in 10 mM DMSO and diluted in HMI-9 to a 40 μM solution (0.4% DMSO). The assay solution was further used to perform serial dilutions (1:2) in a 96-well plate. Mid log bloodstream forms (100 μL) were added in complete HMI-9 medium at a final cell density of 1 × 104 /mL in a well volume of 200 μL after compound addition, leading to a maximum DMSO concentration of 0.2%. Following incubation for 72 h at 37 °C and 5% CO2, 20 μL of 0.5 mM resazurin solution was added, and plates were further incubated for 4 h under similar conditions. Fluorescence was then measured using a Synergy 2 multimode reader (BioTek) at 540 and 620 nm excitation and emission wavelength, respectively. The efficacy of compounds against L. infantum intracellular amastigotes was determined according to Sereno et al. with slight modifications described in detail in the SI. (85)

Liability Assays

The hERG cardiotoxicity assay was performed using the Invitrogen Predictor hERG fluorescence polarization (FP) assay. A membrane fraction containing hERG (Predictor hERG membrane) was used together with a red fluorescent high-affinity ligand of the hERG channel (Predictor hERG Tracer Red). Displacement of the latter from hERG by binding of the test compound can be determined in an FP-based format. (45)
Cytochrome P450 (CYP450) assays against isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 were performed using the Promega P450-Glo assay platform. Microsomal preparations of cytochrome P450s from baculovirus-infected insect cells were used. In this assay, light is generated when a CYP450 enzyme acts on its substrate and a decrease thereof was indicative of inhibitory effects of the tested compound on the respective isoform. (45)
For monitoring mitochondrial toxicity caused by the test compounds in the 786-O cell line, uptake of MitoTracker Red (chloromethyl-X-rosamine) combined with high content imaging was used. Cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium containing 2 mM glutamine, FCS (10% v/v), streptomycin (100 μg/mL), and penicillin G (100 U/mL). (45)
The cytotoxicity assay against A549 cells was performed using the CellTiter-Glo assay from Promega. The number of viable cells present is directly proportional to the cellular ATP content, which is detected. The A549 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and grown in Dulbecco’s modified Eagle medium (DMEM) with FCS (10% v/v), streptomycin (100 μg/mL), and penicillin G (100 U/mL). (45)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00232.

  • Supplemental figures S1–9, supplemental tables S1–12, supplemental experimental procedures and compound characterization, NMR spectra of compounds (PDF)

  • SMILES and activities of compounds (CSV)

Accession Codes

Crystal structures described in this paper are available in the Protein Data Bank with identifiers: 6rx5 (TbPTR1-NADPH/NADP+-1b), 6rx0 (TbPTR1-NADPH/NADP+-2a), 6rx6 (TbPTR1-NADPH/NADP+-2e), 6rxc (LmPTR1-NADPH/NADP+-2e).

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
    • Maria P. Costi - Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy;  Orcidhttps://orcid.org/0000-0002-0443-5402;  Email: [email protected]
    • Alberto Venturelli - Tydock Pharma srl, Strada Gherbella 294/B, 41126 Modena, Italy;  Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy;  Email: [email protected]
    • Rebecca C. Wade - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany;  Faculty of Biosciences, Heidelberg University, D-69120 Heidelberg, Germany;  Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, D-69120 Heidelberg, Germany;  Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, D-69120 Heidelberg, Germany;  Orcidhttps://orcid.org/0000-0001-5951-8670;  Email: [email protected]
  • Authors
    • Ina Pöhner - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany;  Faculty of Biosciences, Heidelberg University, D-69120 Heidelberg, Germany;  Present Address: School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland. (I.P.);  Orcidhttps://orcid.org/0000-0002-2801-8902
    • Antonio Quotadamo - Tydock Pharma srl, Strada Gherbella 294/B, 41126 Modena, Italy;  Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, 41121 Modena, Italy
    • Joanna Panecka-Hofman - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), D-69118 Heidelberg, Germany;  Faculty of Physics, University of Warsaw, 02-093 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0003-4149-9090
    • Rosaria Luciani - Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
    • Matteo Santucci - Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
    • Pasquale Linciano - Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy
    • Giacomo Landi - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
    • Flavio Di Pisa - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
    • Lucia Dello Iacono - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
    • Cecilia Pozzi - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy;  Orcidhttps://orcid.org/0000-0003-2574-3911
    • Stefano Mangani - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy;  Orcidhttps://orcid.org/0000-0003-4824-7478
    • Sheraz Gul - Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
    • Gesa Witt - Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
    • Bernhard Ellinger - Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany
    • Maria Kuzikov - Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, D-22525 Hamburg, Germany;  Orcidhttps://orcid.org/0000-0001-8771-1865
    • Nuno Santarem - Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology, Universidade do Porto, 4200-135 Porto, Portugal
    • Anabela Cordeiro-da-Silva - Instituto de Investigação e Inovação em Saúde, Institute for Molecular and Cell Biology, Universidade do Porto, 4200-135 Porto, Portugal;  Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
  • Author Contributions

    I.P. and A.Q. are joint first authors.

    Author Contributions

    Conceptualization, M.P.C., A.V., R.C.W.; computational methodology and investigation, I.P., J.P.-H.; chemical synthesis methodology and investigation, A.Q.; enzyme assay methodology and investigation, R.L., M.S., P.L.; crystallography methodology and investigation, G.L., F.D.P., L.D.I., C.P.; ADMET methodology and investigation, S.G., G.W., B.E., M.K.; parasite assay methodology and investigation, N.S.; writing – original draft, I.P., writing – review & editing, I.P., J.P.-H., M.P.C., R.C.W.; supervision, S.M., A.C.S., M.P.C., A.V., R.C.W.

  • Notes
    The authors declare no competing financial interest.

    Additional supplementary data are freely available at https://doi.org/10.15490/fairdomhub.1.investigation.417.1: QikProp prediction results for synthesized and in silico pteridines and corresponding SOP. PAINS filtering results, Python modules for correlating QikProp data with experimental activities and for computing a multiple correlation between target and parasite inhibition. Compound library construction data and SOP, prepared docking receptors (PDB) with SOP, all Glide XP rigid-body docking results as PDB files of the receptor–ligand complexes and SOP as well as selected discussed induced fit docking results and corresponding SOP.

Acknowledgments

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This work has received funding from the European Union’s Seventh Framework Programme for research, technological development, and demonstration under grant agreement no. 603240 (NMTrypI, New Medicines for Trypanosomatidic Infections, https://fp7-nmtrypi.eu/). We thank Prof. Antonio Carta, University of Sassari, for providing the reference compounds 1d–1h. I.P., J.P.-H., and R.C.W. gratefully acknowledge the support of the Klaus Tschira Foundation. J.P.-H. acknowledges support from the Polish National Science Centre (grant no. 2016/21/D/NZ1/02806), the BIOMS program at the Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, and the Interdisciplinary Centre for Mathematical and Computational Modelling (ICM), University of Warsaw (grant no. G70-13, GB70-11, GA73-25).

Abbreviations Used

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DHFR

dihydrofolate reductase

HAT

human African trypanosomiasis

MTX

methotrexate

NTDs

neglected tropical diseases

PABA

para-amino benzoic acid

PAINS

pan-assay interference compounds

PTR1

pteridine reductase 1

SI

selectivity index

TS

thymidylate synthase

References

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    The trypanosomiases consist of a group of important animal and human diseases caused by parasitic protozoa of the genus Trypanosoma. In sub-Saharan Africa, the final decade of the 20th century witnessed an alarming resurgence in sleeping sickness (human African trypanosomiasis). In South and Central America, Chagas' disease (American trypanosomiasis) remains one of the most prevalent infectious diseases. Arthropod vectors transmit African and American trypanosomiases, and disease containment through insect control programmes is an achievable goal. Chemotherapy is available for both diseases, but existing drugs are far from ideal. The trypanosomes are some of the earliest diverging members of the Eukaryotae and share several biochemical peculiarities that have stimulated research into new drug targets. However, differences in the ways in which trypanosome species interact with their hosts have frustrated efforts to design drugs effective against both species. Growth in recognition of these neglected diseases might result in progress towards control through increased funding for drug development and vector elimination.
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    Blum, J.; Schmid, C.; Burri, C. Clinical aspects of 2541 patients with second stage human African trypanosomiasis. Acta Trop. 2006, 97, 55– 64,  DOI: 10.1016/j.actatropica.2005.08.001
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    Clinical aspects of 2541 patients with second stage human African trypanosomiasis
    Blum Johannes; Schmid Caecilia; Burri Christian
    Acta tropica (2006), 97 (1), 55-64 ISSN:0001-706X.
    The clinical symptoms and signs of patients with second stage HAT are described for a large cohort of patients treated in a prospective multicentre, multinational study. Special emphasis is given to the influence of disease stage (duration, number of WBC in CSF) and patient age to the clinical picture. Even though the frequencies of symptoms and signs are highly variable between centres, the clinical picture of the disease is similar for all countries. Headache (78.7%), sleeping disorder (74.4%) and lymphadenopathy (56.1%) are the most frequent symptoms and signs and they are similar for all stages of the disease. Lymphadenopathy tends to be highest in the advanced second stage (59.0%). The neurological and psychiatric symptoms increase significantly with the number of WBC in the CSF indicating the stage of progression of the disease. Pruritus is observed in all stages and increases with the number of WBC in CSF from 30 to 55%. In children younger than 7 years, lymphadenopathy is less frequently reported (11.8-37.3%) than in older children or adults (56.4-61.2%). Fever is most frequently reported in children between 2 and 14 years of age (26.1-28.7%) and malnutrition is significantly more frequently observed in children of all ages (43-56%) than in adults (23.5%).
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    Stuart, K.; Brun, R.; Croft, S.; Fairlamb, A.; Gürtler, R. E.; McKerrow, J.; Reed, S.; Tarleton, R. Kinetoplastids: related protozoan pathogens, different diseases. J. Clin. Invest. 2008, 118, 1301– 1310,  DOI: 10.1172/JCI33945
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    Kinetoplastids: related protozoan pathogens, different diseases
    Stuart, Ken; Brun, Reto; Croft, Simon; Fairlamb, Alan; Gurtler, Ricardo E.; McKerrow, Jim; Reed, Steve; Tarleton, Rick
    Journal of Clinical Investigation (2008), 118 (4), 1301-1310CODEN: JCINAO; ISSN:0021-9738. (American Society for Clinical Investigation)
    Kinetoplastids are a group of flagellated protozoans that include the species Trypanosoma and Leishmania, which are human pathogens with devastating health and economic effects. The sequencing of the genomes of some of these species has highlighted their genetic relatedness and underlined differences in the diseases that they cause. As we discuss in this Review, steady progress using a combination of mol., genetic, immunol., and clin. approaches has substantially increased understanding of these pathogens and important aspects of the diseases that they cause. Consequently, the paths for developing addnl. measures to control these "neglected diseases" are becoming increasingly clear, and we believe that the opportunities for developing the drugs, diagnostics, vaccines, and other tools necessary to expand the armamentarium to combat these diseases have never been better.
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    Cunningham, A. C. Parasitic adaptive mechanisms in infection by Leishmania. Exp. Mol. Pathol. 2002, 72, 132– 141,  DOI: 10.1006/exmp.2002.2418
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    Parasitic adaptive mechanisms in infection by Leishmania
    Cunningham, Anna C.
    Experimental and Molecular Pathology (2002), 72 (2), 132-141CODEN: EXMPA6; ISSN:0014-4800. (Academic Press)
    A review. Leishmania are a resilient group of intracellular parasites that infect macrophages. The resultant complex of diseases, or leishmaniases, caused by the parasites affect over twelve million people worldwide. Leishmania have developed unique adaptive mechanisms to ensure their survival in the harsh environments faced throughout their life cycle. These parasites must not only contend with the hostile digestive conditions found within the sand fly vector, but they must also avoid destruction by the host immune system while in the bloodstream, before entering the macrophage. To do so, Leishmania express unique lipophosphoglycan (LPG) mols. and the metalloprotease gp63, among other proteins, on their cell surface. To enter the macrophage, Leishmania utilizes a variety of cellular receptors to mediate endocytosis. Once inside the macrophage, Leishmania is protected from phagolysosome degrdn. by a variety of adaptations to inhibit cellular defense mechanisms. These include the inhibition of phagosome-endosome fusion, hydrolytic enzymes, cell signaling pathways, nitric oxide prodn., and cytokine prodn. While other parasites can also infect macrophages, Leishmania is distinctive in that it not only relies on its own defenses to survive and reproduce within the macrophage phagolysosome, but Leishmania also manipulates the host immune response in order to protect itself and to gain entry into the cell. These unique adaptive mechanisms help promote Leishmania survival. (c) 2002 Academic Press.
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    Herwaldt, B. L. Leishmaniasis. Lancet. 1999, 354, 1191– 1199,  DOI: 10.1016/S0140-6736(98)10178-2
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    Leishmaniasis
    Herwaldt B L
    Lancet (London, England) (1999), 354 (9185), 1191-9 ISSN:0140-6736.
    In 1903, Leishman and Donovan separately described the protozoan now called Leishmania donovani in splenic tissue from patients in India with the life-threatening disease now called visceral leishmaniasis. Almost a century later, many features of leishmaniasis and its major syndromes (ie, visceral, cutaneous, and mucosal) have remained the same; but also much has changed. As before, epidemics of this sandfly-borne disease occur periodically in India and elsewhere; but leishmaniasis has also emerged in new regions and settings, for example, as an AIDS-associated opportunistic infection. Diagnosis still typically relies on classic microbiological methods, but molecular-based approaches are being tested. Pentavalent antimony compounds have been the mainstay of antileishmanial therapy for half a century, but lipid formulations of amphotericin B (though expensive and administered parenterally) represent a major advance for treating visceral leishmaniasis. A pressing need is for the technological advances in the understanding of the immune response to leishmania and the pathogenesis of leishmaniasis to be translated into field-applicable and affordable methods for diagnosis, treatment, and prevention of this disease. This paper discusses the pathogenesis and clinical management of leishmaniasis. Leishmaniasis is a vector-borne disease caused by obligate intramacrophage protozoa, characterized by diversity and complexity. It is endemic in areas of the tropics, subtropics, and southern Europe, in settings ranging from rain forests in Americas to deserts in western Asia, and from rural to periurban areas. Several clinical syndromes are categorized under the term leishmaniasis: most notably visceral, cutaneous, and mucosal leishmaniasis, which result from replication of the parasite in macrophages of the mononuclear phagocyte system, dermis, and naso-oro-pharyngeal mucosa, respectively. These syndromes are caused by a total of about 21 leishmanial species, which are transmitted by about 30 species of phlebotomine sandflies. Clinical manifestation of the disease depends on the type of leishmaniasis, which could be life-threatening systemic infection (visceral), chronic skin sores (cutaneous), or dreaded metastatic complications, which causes facial disfigurement (mucosal). Clinical management is directed to prevent death from visceral leishmaniasis and morbidity from cutaneous and mucosal leishmaniasis. Also included in its management is the ongoing research on immunoregulation of leishmaniasis in the understanding of the immune response to intracellular pathogens and rationalizing vaccine development. General principles on the disease diagnosis and treatment are outlined in this paper.
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    Castillo, E.; Dea-Ayuela, M. A.; Bolás-Fernández, F.; Rangel, M.; González-Rosende, M. E. The kinetoplastid chemotherapy revisited: current drugs, recent advances and future perspectives. Curr. Med. Chem. 2010, 17, 4027– 4051,  DOI: 10.2174/092986710793205345
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    The kinetoplastid chemotherapy revisited: current drugs, recent advances and future perspectives
    Castillo, E.; Dea-Ayuela, M. A.; Bolas-Fernandez, F.; Rangel, M.; Gonzalez-Rosende, M. E.
    Current Medicinal Chemistry (2010), 17 (33), 4027-4051CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
    A review. Leishmaniasis, African sleeping sickness and Chagas disease, caused by the kinetoplastid parasites Leisyhmania spp, Trypanosoma brucei and Trypanosoma cruzi. resp., are among the most important parasitic diseases, affecting millions of people and considered to be within the most relevant group of neglected tropical diseases. The main alternative to control such parasitosis is chemotherapy. Nevertheless, the current chemotherapeutic treatments are far from being satisfactory. This review outlines the current understanding of different drugs against leishmaniasis, African sleeping sickness and Chagas disease, their mechanism of action and resistance. Recent approaches in the area of anti-leishmanial and trypanocidal therapies are also enumerated, new modulators from the mode of action, development of new formulations of old drugs, therapeutic switching and "in silico" drug design.
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    Machado-Silva, A.; Goulart Guimarães, P. P.; Pereira Tavares, C. A.; Sinisterra, R. D. New perspectives for leishmaniasis chemotherapy over current anti-leishmanial drugs: a patent landscape. Expert Opin. Ther. Pat. 2015, 25, 247– 260,  DOI: 10.1517/13543776.2014.993969
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    New perspectives for leishmaniasis chemotherapy over current anti-leishmanial drugs: a patent landscape
    Machado-Silva, Alice; Guimaraes, Pedro Pires Goulart; Tavares, Carlos Alberto Pereira; Sinisterra, Ruben Dario
    Expert Opinion on Therapeutic Patents (2015), 25 (3), 247-260CODEN: EOTPEG; ISSN:1354-3776. (Informa Healthcare)
    Introduction: Although leishmaniasis is estd. to cause the ninth largest disease burden among individual infectious diseases, it is still one of the most neglected diseases in terms of drug development. Current drugs are highly toxic, resistance is common and compliance of patients to treatment is low, as treatment is long and drug price is high. Areas covered: In this review, the authors carried out a patent landscape in search for new perspectives for leishmaniasis therapy. This search encompassed patent documents having priority date between 1994 and 2014. Selected compds. were compared to current anti-leishmanial drugs regarding efficacy and toxicity, when exptl. data were available. Expert opinion: Most patents related to drugs for leishmaniasis have not been produced by the pharmaceutical industry but rather by public research institutes or by universities, and the majority of the inventions disclosed are still in preclin. phase. There is an urgent need to find new ways of funding research for leishmaniasis drugs, incentivizing product development partnerships and pushing forward innovation.
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    Gilbert, I. H. Drug discovery for neglected diseases: molecular target-based and phenotypic approaches. J. Med. Chem. 2013, 56, 7719– 7726,  DOI: 10.1021/jm400362b
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    Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic Approaches
    Gilbert, Ian H.
    Journal of Medicinal Chemistry (2013), 56 (20), 7719-7726CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Drug discovery for neglected tropical diseases is carried out using both target-based and phenotypic approaches. In this paper, target-based approaches are discussed, with a particular focus on human African trypanosomiasis. Target-based drug discovery can be successful, but careful selection of targets is required. There are still very few fully validated drug targets in neglected diseases, and there is a high attrition rate in target-based drug discovery for these diseases. Phenotypic screening is a powerful method in both neglected and non-neglected diseases and has been very successfully used. Identification of mol. targets from phenotypic approaches can be a way to identify potential new drug targets.
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    Müller, J.; Hemphill, A. Drug target identification in protozoan parasites. Expert Opin. Drug Discovery 2016, 11, 815– 824,  DOI: 10.1080/17460441.2016.1195945
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    Borsari, C.; Quotadamo, A.; Ferrari, S.; Venturelli, A.; Cordeiro-da-Silva, A.; Santarem, N.; Costi, M. P. Chapter Two - Scaffolds and biological targets avenue to fight against drug resistance in leishmaniasis. Annu. Rep. Med. Chem. 2018, 51, 39– 95
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    Scaffolds and Biological Targets Avenue to Fight Against Drug Resistance In Leishmaniasis
    Borsari, Chiara; Quotadamo, Antonio; Ferrari, Stefania; Venturelli, Alberto; Cordeiro-da-Silva, Anabela; Santarem, Nuno; Costi, Maria Paola
    Annual Reports in Medicinal Chemistry (2018), 51 (Neglected Diseases: Extensive Space for Modern Drug Discovery), 39-95CODEN: ARMCBI; ISSN:0065-7743. (Elsevier Inc.)
    A review. This chapter is focused on the available knowledge regarding drug resistance in the field of antiparasitic drugs against leishmaniasis. Anti-leishmenia drugs including liposomal amphotericin B, pentamidine-isethionate, miltefosine, antimonials such as meglumine antimoniate and sodium stibogluconate were studied. This work collected scaffolds and biol. targets that are usable for fighting drug resistance in Leishmania infections. This approach could provide drugs suitable for combination therapy, because they may prevent the overexpression of those targets that are involved in the resistance effects.
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    Shuvalov, O.; Petukhov, A.; Daks, A.; Fedorova, O.; Vasileva, E.; Barlev, N. A. One-carbon metabolism and nucleotide biosynthesis as attractive targets for anticancer therapy. Oncotarget 2017, 8, 23955– 23977,  DOI: 10.18632/oncotarget.15053
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    One-carbon metabolism and nucleotide biosynthesis as attractive targets for anticancer therapy
    Shuvalov Oleg; Petukhov Alexey; Daks Alexandra; Fedorova Olga; Vasileva Elena; Barlev Nickolai A; Petukhov Alexey
    Oncotarget (2017), 8 (14), 23955-23977 ISSN:.
    Cancer-related metabolism has recently emerged as one of the "hallmarks of cancer". It has several important features, including altered metabolism of glucose and glutamine. Importantly, altered cancer metabolism connects different biochemical pathways into the one fine-tuned metabolic network, which stimulates high proliferation rates and plasticity to malignant cells. Among the keystones of cancer metabolism are one-carbon metabolism and nucleotide biosynthesis, which provide building blocks to anabolic reactions. Accordingly, the importance of these metabolic pathways for anticancer therapy has well been documented by more than fifty years of clinical use of specific metabolic inhibitors - methotrexate and nucleotides analogs. In this review we discuss one-carbon metabolism and nucleotide biosynthesis as common and specific features of many, if not all, tumors. The key enzymes involved in these pathways also represent promising anti-cancer therapeutic targets. We review different aspects of these metabolic pathways including their biochemistry, compartmentalization and expression of the key enzymes and their regulation at different levels. We also discuss the effects of known inhibitors of these pathways as well as the recent data on other enzymes of the same pathways as perspective pharmacological targets.
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    Anderson, A. C.; Wright, D. L. Antifolate agents: a patent review (2010 - 2013). Expert Opin. Ther. Pat. 2014, 24, 687– 697,  DOI: 10.1517/13543776.2014.898062
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    Antifolate agents: a patent review (2010 - 2013)
    Anderson, Amy C.; Wright, Dennis L.
    Expert Opinion on Therapeutic Patents (2014), 24 (6), 687-697CODEN: EOTPEG; ISSN:1354-3776. (Informa Healthcare)
    A review. The folate biosynthetic pathway, responsible for the de novo synthesis of thymidine and other key cellular components, is essential in all life forms and is esp. crit. in rapidly proliferating cells. As such, druggable targets along this pathway offer opportunities to impact many disease states such as cancer, infectious disease and autoimmune disease. In this article, recent progress on the development of antifolate compds. is reviewed. The evaluation of the patent literature during the period 2010 - 2013 focused on any compds. inhibiting recognized targets on the folate biosynthetic pathway. The folate pathway constitutes a well-validated and well-characterized set of targets; this pathway continues to elicit considerable enthusiasm for new drug discovery from both academic and industrial pharmaceutical research groups. Within the pathway, the enzymes dihydrofolate reductase and thymidylate synthase persist as the most attractive targets for new drug discovery for the treatment of cancer and infectious disease. Importantly, new potential targets for antifolates such as those on the purine biosynthetic pathway have been recently explored. The use of structure-based drug design is a major aspect in modern approaches to these drug targets.
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    Hawser, S.; Lociuro, S.; Islam, K. Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol. 2006, 71, 941– 948,  DOI: 10.1016/j.bcp.2005.10.052
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    Dihydrofolate reductase inhibitors as antibacterial agents
    Hawser, Stephen; Lociuro, Sergio; Islam, Khalid
    Biochemical Pharmacology (2006), 71 (7), 941-948CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
    A review. Although only a few DHFR inhibitors have progressed as antibiotics to the market there is much renewed interest in the discovery and development of new generation DHFR inhibitors as antibacterial agents. This article describes the success in exploiting DHFR as a drugable target as exemplified by trimethoprim (TMP) and the development of several new diaminopyrimidines. Iclaprim, a recent example of a novel diaminopyrimidine currently in Phase III clin. trials, is also described together with several examples of anti-DHFR antibacterial compds. in pre-clin. development.
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    Yuthavong, Y.; Yuvaniyama, J.; Chitnumsub, P.; Vanichtanankul, J.; Chusacultanachai, S.; Tarnchompoo, B.; Vilaivan, T.; Kamchonwongpaisan, S. Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors. Parasitology 2005, 130, 249– 259,  DOI: 10.1017/S003118200400664X
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    Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors
    Yuthavong, Y.; Yuvaniyama, J.; Chitnumsub, P.; Vanichtanankul, J.; Chusacultanachai, S.; Tarnchompoo, B.; Vilaivan, T.; Kamchonwongpaisan, S.
    Parasitology (2005), 130 (3), 249-259CODEN: PARAAE; ISSN:0031-1820. (Cambridge University Press)
    A review. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific addnl. sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes assocd. with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent antimalarial activities against drug-resistant P. falciparum bearing the mutant enzymes.
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    Christensen, K. E.; MacKenzie, R. E. Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals. Bioessays 2006, 28, 595– 605,  DOI: 10.1002/bies.20420
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    Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals
    Christensen, Karen E.; MacKenzie, Robert E.
    BioEssays (2006), 28 (6), 595-605CODEN: BIOEEJ; ISSN:0265-9247. (John Wiley & Sons, Inc.)
    A review. In eukaryotes, folate metab. is compartmentalized between the cytoplasm and organelles. The folate pathways of mitochondria are adapted to serve the metab. of the organism. In yeast, mitochondria support cytoplasmic purine synthesis through the generation of formate. This pathway is important but not essential for survival, consistent with the flexibility of yeast metab. In plants, the mitochondrial pathways support photorespiration by generating serine from glycine. This pathway is essential under photosynthetic conditions and the enzyme expression varies with photosynthetic activity. In mammals, the expression of the mitochondrial enzymes varies in tissues and during development. In embryos, mitochondria supply formate and glycine for purine synthesis, a process essential for survival; in adult tissues, flux through mitochondria can favor serine prodn. Thus, the differences in the folate pathways of mitochondria depending on species, tissues and developmental stages, profoundly alter the nature of their metabolic contribution.
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    Cullia, G.; Tamborini, L.; Conti, P.; De Micheli, C.; Pinto, A. Folates in Trypanosoma brucei: Achievements and opportunities. ChemMedChem. 2018, 13, 2150– 2158,  DOI: 10.1002/cmdc.201800500
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    Folates in Trypanosoma brucei: Achievements and Opportunities
    Cullia, Gregorio; Tamborini, Lucia; Conti, Paola; De Micheli, Carlo; Pinto, Andrea
    ChemMedChem (2018), 13 (20), 2150-2158CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Trypanosoma brucei is the agent of human African trypanosomiasis (HAT), a neglected disease that threatens the lives of 65 million people in sub-Saharan Africa every year. Unfortunately, available therapies are unsatisfactory, due primarily to safety issues and development of drug resistance. Over the last decades significant effort has been made in the discovery of new potential anti-HAT agents, with help from the World Health Organization (WHO) and private-public partnerships such as the Drugs for Neglected Diseases Initiative (DNDi). Whereas antifolates have been a valuable source of drugs against bacterial infections and malaria, compds. effective against T. brucei have not yet been identified. Considering the relatively simple folate metabolic pathway in T. brucei, along with results obtained in this research field so far, we believe that further investigations might lead to effective chemotherapeutic agents. Herein we present a selection of the more promising results obtained so far in this field, underlining the opportunities that could lead to successful therapeutic approaches in the future.
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    Bello, A. R.; Nare, B.; Freedman, D.; Hardy, L.; Beverley, S. M. PTR1: A reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11442– 11446,  DOI: 10.1073/pnas.91.24.11442
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    19
    PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major
    Bello, Alexandre R.; Nare, Bakela; Freedman, Daniel; Hardy, Larry; Beverley, Stephen M.
    Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (24), 11442-6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Trypanosomatid protozoans are pterin auxotrophs, a finding noted decades ago which heralded the discovery of key metabolic roles played by pteridines in eukaryotes. We have now identified the enzyme mediating unconjugated pteridine salvage in the human parasite Leishmania major. PTR1 is the gene in the amplified H region responsible for methotrexate (MTX) resistance, and belongs to a large family of oxidoreductases with diverse substrates and roles. We generated Leishmania lacking PTR1 by homologous gene targeting, and these ptr1- mutants required reduced biopterin (dihydro- or tetrahydrobiopterin) for growth. PTR1 purified from engineered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biopterin reductase activity. PTR1 showed good activity with folate and significant activity with dihydrofolate and dihydrobiopterin, but not with quinonoid dihydrobiopterin. PTR1 thus differs considerably from previously reported pteridine reductases of trypanosomatids and vertebrates. Pteridine reductase activity was diminished in ptr1- Leishmania and was evaluated in transfected parasites bearing multiple copies of PTR1; correspondingly, ptr1- was MTX-hypersensitive whereas the multicopy transfectant was MTX-resistant. The concordance of the biochem. and genetic properties of PTR1 suggests that this is the primary enzyme mediating pteridine salvage. These findings suggest several possible mechanisms for PTR1-mediated MTX resistance and should aid in the design of rational chemotherapy.
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  20. 20
    Dawson, A.; Gibellini, F.; Sienkiewicz, N.; Tulloch, L. B.; Fyfe, P. K.; McLuskey, K.; Fairlamb, A. H.; Hunter, W. N. Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate. Mol. Microbiol. 2006, 61, 1457– 1468,  DOI: 10.1111/j.1365-2958.2006.05332.x
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    20
    Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate
    Dawson, Alice; Gibellini, Federica; Sienkiewicz, Natasha; Tulloch, Lindsay B.; Fyfe, Paul K.; McLuskey, Karen; Fairlamb, Alan H.; Hunter, William N.
    Molecular Microbiology (2006), 61 (6), 1457-1468CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
    The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor detd. at 2.2 Å resoln. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic center are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chem. properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
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  21. 21
    Vickers, T. J.; Beverley, S. M. Folate metabolic pathways in Leishmania. Essays Biochem. 2011, 51, 63– 80,  DOI: 10.1042/bse0510063
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    Folate metabolic pathways in Leishmania
    Vickers, Tim J.; Beverley, Stephen M.
    Essays in Biochemistry (2011), 51 (Molecular Parasitology), 63-80CODEN: ESBIAV; ISSN:0071-1365. (Portland Press Ltd.)
    A review. Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metab. from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compds. developed originally for treatment of human cancers or other infectious agents.
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  22. 22
    Ong, H. B.; Sienkiewicz, N.; Wyllie, S.; Fairlamb, A. H. Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major. J. Biol. Chem. 2011, 286, 10429– 10438,  DOI: 10.1074/jbc.M110.209593
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    22
    Dissecting the Metabolic Roles of Pteridine Reductase 1 in Trypanosoma brucei and Leishmania major
    Ong, Han B.; Sienkiewicz, Natasha; Wyllie, Susan; Fairlamb, Alan H.
    Journal of Biological Chemistry (2011), 286 (12), 10429-10438CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H4B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity assocd. with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiol. pH and ionic strength and compared with LmQDPR. Specificity consts. (kcat/Km) for LmPTR1 are similar with dihydrobiopterin (H2B) and quinonoid dihydrobiopterin (qH2B) as substrates and about 20-fold lower than LmQDPR with qH2B. In contrast, TbPTR1 shows a 10-fold higher kcat/Km for H2B over qH2B. Anal. of Trypanosoma brucei isolated from infected rats revealed that H4B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H4B. Gene knock-out expts. confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H4B as a medium supplement. These cells grew normally with H4B, which spontaneously oxidizes to qH2B, but were unable to survive in the absence of pterin or with either biopterin or H2B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metab. in African trypanosomes and underline its potential as a drug target.
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    Sienkiewicz, N.; Ong, H. B.; Fairlamb, A. H. Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice. Mol. Microbiol. 2010, 77, 658– 671,  DOI: 10.1111/j.1365-2958.2010.07236.x
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    23
    Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice
    Sienkiewicz, Natasha; Ong, Han B.; Fairlamb, Alan H.
    Molecular Microbiology (2010), 77 (3), 658-671CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
    Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metab. in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addn. of tetrahydrobiopterin to cultures. Loss of PTR1 was assocd. with gross morphol. changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased nos. of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localization to the cytosol. Mis-localization was confirmed by digitonin fractionation expts. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.
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  24. 24
    Mpamhanga, C. P.; Spinks, D.; Tulloch, L. B.; Shanks, E. J.; Robinson, D. A.; Collie, I. T.; Fairlamb, A. H.; Wyatt, P. G.; Frearson, J. A.; Hunter, W. N.; Gilbert, I. H.; Brenk, R. One scaffold, three binding modes: Novel and selective pteridine reductase 1 inhibitors derived from fragment hits discovered by virtual screening. J. Med. Chem. 2009, 52, 4454– 4465,  DOI: 10.1021/jm900414x
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    24
    One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening
    Mpamhanga, Chidochangu P.; Spinks, Daniel; Tulloch, Lindsay B.; Shanks, Emma J.; Robinson, David A.; Collie, Iain T.; Fairlamb, Alan H.; Wyatt, Paul G.; Frearson, Julie A.; Hunter, William N.; Gilbert, Ian H.; Brenk, Ruth
    Journal of Medicinal Chemistry (2009), 52 (14), 4454-4465CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The enzyme pteridine reductase 1 (PTR1) is a potential target for new compds. to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chem. series were identified contg. aminobenzothiazole and aminobenzimidazole scaffolds, resp. One of the hits (2-amino-5-chlorobenzimidazole) was subjected to crystal structure anal. and a high resoln. crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogs (2-aminobenzimidazole and 1-(3,4-dichlorobenzyl)-2-aminobenzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein-ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichlorobenzyl)-2-aminobenzimidazole, derivs. were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochem. properties were obtained.
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  25. 25
    Spinks, D.; Ong, H. B.; Mpamhanga, C. P.; Shanks, E. J.; Robinson, D. A.; Collie, I. T.; Read, K. D.; Frearson, J. A.; Wyatt, P. G.; Brenk, R.; Fairlamb, A. H.; Gilbert, I. H. Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1. ChemMedChem. 2011, 6, 302– 308,  DOI: 10.1002/cmdc.201000450
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    25
    Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase 1
    Spinks, Daniel; Ong, Han B.; Mpamhanga, Chidochangu P.; Shanks, Emma J.; Robinson, David A.; Collie, Iain T.; Read, Kevin D.; Frearson, Julie A.; Wyatt, Paul G.; Brenk, Ruth; Fairlamb, Alan H.; Gilbert, Ian H.
    ChemMedChem (2011), 6 (2), 302-308CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimization of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivs. Data are reported on 33 compds. This series was initially discovered by a virtual screening campaign. The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chem. and structure-based approaches, we were able to derive compds. with potent activity against T. brucei PTR1 (Kappi = 7 nM), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compds. displayed weak activity against the parasites. Kinetic studies and anal. indicate that the main reason for the lack of cell potency is due to the compds. having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km = 25 nM and Ki = 2.3 nM, resp.).
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  26. 26
    Cavazzuti, A.; Paglietti, G.; Hunter, W. N.; Gamarro, F.; Piras, S.; Loriga, M.; Allecca, S.; Corona, P.; McLuskey, K.; Tulloch, L.; Gibellini, F.; Ferrari, S.; Costi, M. P. Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1448– 1453,  DOI: 10.1073/pnas.0704384105
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    26
    Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development
    Cavazzuti, Antonio; Paglietti, Giuseppe; Hunter, William N.; Gamarro, Francisco; Piras, Sandra; Loriga, Mario; Alleca, Sergio; Corona, Paola; McLuskey, Karen; Tulloch, Lindsay; Gibellini, Federica; Ferrari, Stefania; Costi, Maria Paola
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (5), 1448-1453CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, a rapid-screening strategy using a folate-based library was combined with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling detd. selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivs., and nine compds. showed greater activity against parasite enzymes compared with human enzymes. Compd. I [R = H, Me (II)] displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:I ternary complex revealed a substrate-like binding mode distinct from that previously obsd. for similar compds. A second round of design, synthesis, and assay produced a compd. II with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also detd. Biol. evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was obsd. when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a redn. in toxicity of treatment was obsd. with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.
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    Ivanetich, K. M.; Santi, D. V. Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa. FASEB J. 1990, 4, 1591– 1597,  DOI: 10.1096/fasebj.4.6.2180768
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    Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa
    Ivanetich, Kathryn M.; Santi, Daniel V.
    FASEB Journal (1990), 4 (6), 1591-7CODEN: FAJOEC; ISSN:0892-6638.
    A review with 50 refs. on the structure and function of the title enzymes in Leishmania, Plasmodium, and other protozoa.
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    Schormann, N.; Senkovich, O.; Walker, K.; Wright, D. L.; Anderson, A. C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function. Proteins 2008, 73, 889– 901,  DOI: 10.1002/prot.22115
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    28
    Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function
    Schormann, N.; Senkovich, O.; Walker, K.; Wright, D. L.; Anderson, A. C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D.
    Proteins: Structure, Function, and Bioinformatics (2008), 73 (4), 889-901CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    We have employed a structure-based three-dimensional quant. structure-activity relationship (3D-QSAR) approach to predict the biochem. activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between exptl. and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compds. (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of addnl. activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.
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    Schormann, N.; Velu, S. E.; Murugesan, S.; Senkovich, O.; Walker, K.; Chenna, B. C.; Shinkre, B.; Desai, A.; Chattopadhyay, D. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase. Bioorg. Med. Chem. 2010, 18, 4056– 4066,  DOI: 10.1016/j.bmc.2010.04.020
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    29
    Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase
    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish
    Bioorganic & Medicinal Chemistry (2010), 18 (11), 4056-4066CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas' disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compd. was detd. against T. cruzi and human DHFR. One of these compds., Et 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystd. with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was detd. Mol. docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compds. are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the resp. enzymes were calcd. based on the exptl. or docked binding mode. An estd. 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.
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  30. 30
    Corona, P.; Gibellini, F.; Cavalli, A.; Saxena, P.; Carta, A.; Loriga, M.; Luciani, R.; Paglietti, G.; Guerrieri, D.; Nerini, E.; Gupta, S.; Hannaert, V.; Michels, P. A. M.; Ferrari, S.; Costi, P. M. Structure-based selectivity optimization of piperidine-pteridine derivatives as potent Leishmania pteridine reductase inhibitors. J. Med. Chem. 2012, 55, 8318– 8329,  DOI: 10.1021/jm300563f
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    30
    Structure-Based Selectivity Optimization of Piperidine-Pteridine Derivatives as Potent Leishmania Pteridine Reductase Inhibitors
    Corona, Paola; Gibellini, Federica; Cavalli, Andrea; Saxena, Puneet; Carta, Antonio; Loriga, Mario; Luciani, Rosaria; Paglietti, Giuseppe; Guerrieri, Davide; Nerini, Erika; Gupta, Shreedhara; Hannaert, Veronique; Michels, Paul A. M.; Ferrari, Stefania; Costi, Paola M.
    Journal of Medicinal Chemistry (2012), 55 (19), 8318-8329CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compd. library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was obsd. for compd. 5b (I). This work represents a step forward in the identification of effective antileishmania agents.
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    Panecka-Hofman, J.; Pöhner, I.; Spyrakis, F.; Zeppelin, T.; Di Pisa, F.; Dello Iacono, L.; Bonucci, A.; Quotadamo, A.; Venturelli, A.; Mangani, S.; Costi, M. P.; Wade, R. C. Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 3215– 3230,  DOI: 10.1016/j.bbagen.2017.09.012
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    31
    Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors
    Panecka-Hofman, Joanna; Poehner, Ina; Spyrakis, Francesca; Zeppelin, Talia; Di Pisa, Flavio; Dello Iacono, Lucia; Bonucci, Alessio; Quotadamo, Antonio; Venturelli, Alberto; Mangani, Stefano; Costi, Maria Paola; Wade, Rebecca C.
    Biochimica et Biophysica Acta, General Subjects (2017), 1861 (12), 3215-3230CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
    Multi-target approaches are necessary to properly analyze or modify the function of a biochem. pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of exptl. validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter. The authors computationally compared the structural, dynamic and physico-chem. properties of the targets. The authors based the anal. on available inhibitory activity and crystallog. data, including a crystal structure of the bifunctional T. cruzi DHFR-TS with tetrahydrofolate bound detd. Due to the low sequence and structural similarity of the targets analyzed, the authors employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. The authors' anal. provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallog. structures. The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosomatid agents. Apart from the target-based discovery of selective compds., the authors' approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.
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  32. 32
    Tulloch, L. B.; Martini, V. P.; Iulek, J.; Huggan, J. K.; Lee, J. H.; Gibson, C. L.; Smith, T. K.; Suckling, C. J.; Hunter, W. N. Structure-based design of pteridine reductase inhibitors targeting African sleeping sickness and the leishmaniases. J. Med. Chem. 2010, 53, 221– 229,  DOI: 10.1021/jm901059x
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    Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the Leishmaniasis
    Tulloch, Lindsay B.; Martini, Viviane P.; Iulek, Jorge; Huggan, Judith K.; Lee, Jeong Hwan; Gibson, Colin L.; Smith, Terry K.; Suckling, Colin J.; Hunter, William N.
    Journal of Medicinal Chemistry (2010), 53 (1), 221-229CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. The authors adopted a structure-based approach to the design of novel PTR1 inhibitors based on three mol. scaffolds. A series of compds., most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compds. displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a mol. bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. The authors tested two new compds. with known DHFR inhibitors. A synergistic effect was obsd. for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.
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    Quotadamo, A.; Linciano, P.; Costi, M. P.; Venturelli, A. Optimization of N-alkylation in the synthesis of methotrexate and pteridine-based derivatives under microwave-irradiation. ChemistrySelect 2019, 4, 4429– 4433,  DOI: 10.1002/slct.201900721
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    Optimization of N-alkylation in the Synthesis of Methotrexate and Pteridine-based Derivatives Under Microwave-Irradiation
    Quotadamo, Antonio; Linciano, Pasquale; Costi, Maria Paola; Venturelli, Alberto
    ChemistrySelect (2019), 4 (15), 4429-4433CODEN: CHEMUD; ISSN:2365-6549. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A new efficient and improved microwave-assisted lab-scale process for the prepn. of I and congeners II [R = Et, Ph, benzyl, etc.; R1 = H, Et, Ph, benzyl; RR1 = N-morpholinyl, 4-benzylpiperazin-1-yl] were described. Starting from the com. available 2,4-diamino-6-(hydroxymethyl)pteridine (Pt-OH), I was obtained with an overall 94% yield through a three steps procedure. The crucial yield-limiting and time-consuming step of SN2 substitution between halogenated pteridine and nucleophilic arom. amine was taken. The innovative process, conducted under microwave irradn., improved yield and purity and in particular reduced the reaction time from days to 20 min. The optimized protocol was successfully applied to the synthesis of diverse pteridine-based derivs. II and to the prepn. in gram-scale of antiparasitic MTX derivs. I for in-vivo studies. This new optimized synthetic procedure therefore represented a worthy alternative to the current protocols for the prepn. of pteridine-based derivs.
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    Sajiki, H.; Ikawa, T.; Hirota, K. Reductive and catalytic monoalkylation of primary amines using nitriles as an alkylating reagent. Org. Lett. 2004, 6, 4977– 4980,  DOI: 10.1021/ol047871o
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    Reductive and catalytic monoalkylation of primary amines using nitriles as an alkylating reagent
    Sajiki, Hironao; Ikawa, Takashi; Hirota, Kosaku
    Organic Letters (2004), 6 (26), 4977-4980CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
    A selective and catalytic mono-N-alkylation method of both arom. and aliph. amines using nitriles as an alkylating agent with Pd/C or Rh/C as a catalyst is described. This method was particularly attractive to provide an environmentally benign and applicable alkylation method of amines without using toxic and corrosive alkylating agents such as alkyl halides and carbonyl compds.
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    Ikawa, T.; Fujita, Y.; Mizusaki, T.; Betsuin, S.; Takamatsu, H.; Maegawa, T.; Monguchi, Y.; Sajiki, H. Selective N-alkylation of amines using nitriles under hydrogenation conditions: facile synthesis of secondary and tertiary amines. Org. Biomol. Chem. 2012, 10, 293– 304,  DOI: 10.1039/C1OB06303K
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    Selective N-alkylation of amines using nitriles under hydrogenation conditions: facile synthesis of secondary and tertiary amines
    Ikawa, Takashi; Fujita, Yuki; Mizusaki, Tomoteru; Betsuin, Sae; Takamatsu, Haruki; Maegawa, Tomohiro; Monguchi, Yasunari; Sajiki, Hironao
    Organic & Biomolecular Chemistry (2012), 10 (2), 293-304CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Nitriles were found to be highly effective alkylating reagents for the selective N-alkylation of amines under catalytic hydrogenation conditions. For the arom. primary amines, the corresponding secondary amines were selectively obtained under Pd/C-catalyzed hydrogenation conditions. Although the use of electron poor arom. amines or bulky nitriles showed a lower reactivity toward the reductive alkylation, the addn. of NH4OAc enhanced the reactivity to give secondary arom. amines in good to excellent yields. Under the same reaction conditions, arom. nitro compds. instead of the arom. primary amines could be directly transformed into secondary amines via a domino reaction involving the one-pot hydrogenation of the nitro group and the reductive alkylation of the amines. While aliph. amines were effectively converted to the corresponding tertiary amines under Pd/C-catalyzed conditions, Rh/C was a highly effective catalyst for the N-monoalkylation of aliph. primary amines without over-alkylation to the tertiary amines. Furthermore, the combination of the Rh/C-catalyzed N-monoalkylation of the aliph. primary amines and addnl. Pd/C-catalyzed alkylation of the resulting secondary aliph. amines could selectively prep. aliph. tertiary amines possessing three different alkyl groups. According to the mechanistic studies, it seems reasonable to conclude that nitriles were reduced to aldimines before the nucleophilic attack of the amine during the first step of the reaction.
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    Ayedi, M. A.; Le Bigot, Y.; Ammar, H.; Abid, S.; El Gharbi, R.; Delmas, M. Synthesis of primary amines by one-pot reductive amination of aldehydes. Synth. Commun. 2013, 43, 2127– 2133,  DOI: 10.1080/00397911.2012.714830
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    Synthesis of primary amines by one-pot reductive amination of aldehydes
    Ayedi, Mohamed Ali; Le Bigot, Yves; Ammar, Houcine; Abid, Souhir; El Gharbi, Rachid; Delmas, Michel
    Synthetic Communications (2013), 43 (16), 2127-2133CODEN: SYNCAV; ISSN:0039-7911. (Taylor & Francis, Inc.)
    A novel, one-pot, two-step reductive amination of aldehydes for the atom-economical synthesis of primary amines was reported. The amination step was carried out with hydroxylammonium chloride and does not require the use of a base. In the subsequent redn. step, a metal zinc/hydrochloride acid system was used. This method is applicable to both aliph. and arom. aldehydes. The operational simplicity, the short reaction times, and the mild reaction conditions add to the value of this method as a practical alternative to the reductive amination of aldehydes. Go to the publisher's online edition of Synthetic Communications to view the free supplemental file.
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    Gourley, D. G.; Schüttelkopf, A. W.; Leonard, G. A.; Luba, J.; Hardy, L. W.; Beverley, S. M.; Hunter, W. N. Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites. Nat. Struct. Biol. 2001, 8, 521– 525,  DOI: 10.1038/88584
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    Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites
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    Nature Structural Biology (2001), 8 (6), 521-525CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)
    Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step redn. of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structure of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first redn. uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.
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    Di Pisa, F.; Landi, G.; Dello Iacono, L.; Pozzi, C.; Borsari, C.; Ferrari, S.; Santucci, M.; Santarem, N.; Cordeiro-da-Silva, A.; Moraes, C. B.; Alcantara, L. M.; Fontana, V.; Freitas-Junior, L. H.; Gul, S.; Kuzikov, M.; Behrens, B.; Pöhner, I.; Wade, R. C.; Costi, M. P.; Mangani, S. Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity. Molecules 2017, 22, 426,  DOI: 10.3390/molecules22030426
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    Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity
    Di Pisa, Flavio; Landi, Giacomo; Iacono, Lucia Dello; Pozzi, Cecilia; Borsari, Chiara; Ferrari, Stefania; Santucci, Matteo; Santarem, Nuno; Cordeiro-da-Silva, Anabela; Moraes, Carolina B.; Alcantara, Laura M.; Fontana, Vanessa; Freitas, Lucio H., Jr.; Gul, Sheraz; Kuzikov, Maria; Behrens, Birte; Pohner, Ina; Wade, Rebecca C.; Costi, Maria Paola; Mangani, Stefano
    Molecules (2017), 22 (3), 426/1-426/16CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
    Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogs (1-3) of previously published chromen-4-one derivs. were synthesized and biol. evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compd. 1 and the first crystal structures of LmPTR1-flavanone complexes (compds. 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivs. was explained by comparison of obsd. and predicted binding modes of the compds. Compd. 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compds. targeting multiple PTR1 variants.
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    Lloyd, M. D. High-Throughput Screening for the Discovery of Enzyme Inhibitors. J. Med. Chem. 2020, 63, 10742– 10772,  DOI: 10.1021/acs.jmedchem.0c00523
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    High-Throughput Screening for the Discovery of Enzyme Inhibitors
    Lloyd, Matthew D.
    Journal of Medicinal Chemistry (2020), 63 (19), 10742-10772CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Enzymes are common targets in high-throughput screening and related campaigns. An anal. of papers published between 1990 and 2018 showed that kinases were the most common enzymes investigated, fluorescence-based assays were the most common readout method, and cancer and bacterial infections were the most common therapeutic areas. High-throughput screening and fragment-screening campaigns published between 2017 and 2019 were analyzed in more depth, giving 75 examples of hit to lead development. Kinases, phosphatases, proteases, and peptidases were the most common targets, fluorescent assays were the most commonly used, and a wide variety of structural features were obsd. within the derived drugs. Hit frequency was largely independent of library size and pos. correlated with Z' value for the assay. Binding of metal ions to library compds. and substrates is an underappreciated source of false-pos. results and unreproducible behavior.
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    Holdgate, G.; Meek, T.; Grimley, R. Mechanistic enzymology in drug discovery: a fresh perspective. Nat. Rev. Drug Discovery 2018, 17, 115– 132,  DOI: 10.1038/nrd.2017.219
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    Mechanistic enzymology in drug discovery: a fresh perspective
    Holdgate, Geoffrey A.; Meek, Thomas D.; Grimley, Rachel L.
    Nature Reviews Drug Discovery (2018), 17 (2), 115-132CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
    A review. Given the therapeutic and com. success of small-mol. enzyme inhibitors, as exemplified by kinase inhibitors in oncol., a major focus of current drug-discovery and development efforts is on enzyme targets. Understanding the course of an enzyme-catalyzed reaction can help to conceptualize different types of inhibitor and to inform the design of screens to identify desired mechanisms. Exploiting this information allows the thorough evaluation of diverse compds., providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. This review highlights the rationale for conducting high-quality mechanistic enzymol. studies and considers the added value in combining such studies with orthogonal biophys. methods.
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    Role of folylpolygutamate synthetase (FPGS) in antifolate chemotherapy; a biochemical and clinical update
    Synold T W; Willits E M; Barredo J C
    Leukemia & lymphoma (1996), 21 (1-2), 9-15 ISSN:1042-8194.
    Even though folate antimetabolites were introduced over forty years ago, they continue to be the backbone of many active chemotherapeutic regimens used by medical and pediatric oncologists. The recognition of polyglutamylation by folylpolyglutamate synthetase (FPGS) as an important metabolic step in the "activation" of classical antifolates and novel drugs aimed at thymidylate synthase (TS) and de novo purine synthesis, has resulted in renewed interest in this class of drugs. In addition, the emergence of secondary neoplasms in patients treated with alkylating agents and topoisomerase inhibitors in contrast to the exceptional safety record of antimetabolites, underscores the need for clinical trials that incorporate new strategies with known active antimetabolites and novel promising agents. In that context, FPGS is an important target for further laboratory and clinical investigations.
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    Gibson, M. W.; Dewar, S.; Ong, H. B.; Sienkiewicz, N.; Fairlamb, A. H. Trypanosoma brucei DHFR-TS revisited: characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase. PLoS Negl. Trop. Dis. 2016, 10, e0004714,  DOI: 10.1371/journal.pntd.0004714
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    Prediction of drug solubility from Monte Carlo simulations
    Jorgensen, William L.; Duffy, Erin M.
    Bioorganic & Medicinal Chemistry Letters (2000), 10 (11), 1155-1158CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
    Monte Carlo statistical mechanics simulations have been carried out for 150 org. solutes in water. Phys. significant descriptors such as the solvent-accessible surface area, nos. of hydrogen bonds, and indexes for cohesive interactions in solids are correlated with pharmacol. important properties including octanol/water partition coeff. (log P) and aq. soly. (log S). The regression equation for log S only requires five descriptors to provide a correlation coeff., r2, of 0.9 and rms error of 0.7 for the 150 solutes. The descriptors can form a basis for structural modifications to guide an analog's properties into desired ranges.
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    Borsari, C.; Luciani, R.; Pozzi, C.; Poehner, I.; Henrich, S.; Trande, M.; Cordeiro-da-Silva, A.; Santarem, N.; Baptista, C.; Tait, A.; Di Pisa, F.; Dello Iacono, L.; Landi, G.; Gul, S.; Wolf, M.; Kuzikov, M.; Ellinger, B.; Reinshagen, J.; Witt, G.; Gribbon, P.; Kohler, M.; Keminer, O.; Behrens, B.; Costantino, L.; Tejera Nevado, P.; Bifeld, E.; Eick, J.; Clos, J.; Torrado, J.; Jiménez-Antón, M. D.; Corral, M. J.; Alunda, J. M.; Pellati, F.; Wade, R. C.; Ferrari, S.; Mangani, S.; Costi, M. P. Profiling of flavonol derivatives for the development of antitrypanosomatidic drugs. J. Med. Chem. 2016, 59, 7598– 7616,  DOI: 10.1021/acs.jmedchem.6b00698
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    Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs
    Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Poehner, Ina; Henrich, Stefan; Trande, Matteo; Cordeiro-da-Silva, Anabela; Santarem, Nuno; Baptista, Catarina; Tait, Annalisa; Di Pisa, Flavio; Dello Iacono, Lucia; Landi, Giacomo; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Reinshagen, Jeanette; Witt, Gesa; Gribbon, Philip; Kohler, Manfred; Keminer, Oliver; Behrens, Birte; Costantino, Luca; TejeraNevado, Paloma; Bifeld, Eugenia; Eick, Julia; Clos, Joachim; Torrado, Juan; Jimenez-Anton, Maria D.; Corral, Maria J.; Alunda, Jose Ma; Pellati, Federica; Wade, Rebecca C.; Ferrari, Stefania; Mangani, Stefano; Costi, Maria Paola
    Journal of Medicinal Chemistry (2016), 59 (16), 7598-7616CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivs. was synthesized. Twelve compds. showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the obsd. structure-activity relationships. Compd. 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compd. 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compd. Combination studies with methotrexate revealed that compd. 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concn. of 1.3 μM, 11.7-fold dose redn. index and no toxicity toward host cells. Our results provide the basis for further chem. modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.
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    Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, O. M.; Ferrari, S.; Marverti, G.; Guerrieri, D.; Ligabue, A.; Frassineti, C.; Pozzi, C.; Mangani, S.; Fessas, D.; Guerrini, R.; Ponterini, G.; Wade, R. C.; Costi, M. P. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, E542– E549,  DOI: 10.1073/pnas.1104829108
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    Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase
    Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; Henrich, Stefan; Salo-Ahen, Outi M. H.; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; Pozzi, Cecilia; Mangani, Stefano; Fessas, Dimitrios; Guerrini, Remo; Ponterini, Glauco; Wade, Rebecca C.; Costi, M. Paola
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (34), E542-E549, SE542/1-SE542/22CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clin. important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concns. in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.
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    Benvenuti, M.; Mangani, S. Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2007, 2, 1633– 1651,  DOI: 10.1038/nprot.2007.198
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    Crystallization of soluble proteins in vapor diffusion for X-ray crystallography
    Benvenuti, Manuela; Mangani, Stefano
    Nature Protocols (2007), 2 (7), 1633-1651CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    The prepn. of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biol. macromol. The complete automation of the crystn. procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. Many research projects from departmental labs. are devoted to the study of few specific proteins. Here, the authors try to provide a series of protocols for the crystn. of sol. proteins, esp. the difficult ones, tailored for small-scale research groups. An est. of the time needed to complete each of the steps described can be found at the end of each section.
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    Battye, T. G. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.; Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol Crystallogr 2011, 67, 271– 281,  DOI: 10.1107/S0907444910048675
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    iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM
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    Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 271-281CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    IMOSFLM is a graphical user interface to the diffraction data-integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but addnl. menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calcn., cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed.
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    Powell, H. R.; Johnson, O.; Leslie, A. G. W. Autoindexing diffraction images with iMosflm. Acta Crystallogr. D Biol Crystallogr 2013, 69, 1195– 1203,  DOI: 10.1107/S0907444912048524
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    Autoindexing diffraction images with iMosflm
    Powell, Harold R.; Johnson, Owen; Leslie, Andrew G. W.
    Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1195-1203CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    A review. An overview of autoindexing diffraction images based on one-dimensional fast Fourier transforms is presented. The implementation of the algorithm in the Mosflm/iMosflm program suite is described with a discussion of practical issues that may arise and ways of assessing the success or failure of the procedure. Recent developments allow indexing of images that show multiple lattices, and several examples demonstrate the success of this approach in real cases.
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    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol Crystallogr 2006, 62, 72– 82,  DOI: 10.1107/S0907444905036693
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    Scaling and assessment of data quality
    Evans, Philip
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    The various phys. factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analyzed to set the real resoln. of the data set, to detect bad regions (e.g. bad images), to analyze radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation anal. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be detd. from scores such as correlation coeffs. between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.
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    Evans, P. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol Crystallogr. 2011, 67, 282– 292,  DOI: 10.1107/S090744491003982X
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    An introduction to data reduction: Space-group determination, scaling and intensity statistics
    Evans, Philip R.
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    A review. This paper presents an overview of how to run the CCP4 programs for data redn. (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers detn. of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examn. of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estn. of |F| from intensity and finally examn. of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups.
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    CCP4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol Crystallogr. 1994, 50, 760– 763,  DOI: 10.1107/S0907444994003112
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    Vagin, A.; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol Crystallogr. 2010, 66, 22– 25,  DOI: 10.1107/S0907444909042589
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    MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.
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    Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol Crystallogr 2011, 67, 355– 367,  DOI: 10.1107/S0907444911001314
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    REFMAC5 for the refinement of macromolecular crystal structures
    Murshudov, Garib N.; Skubak, Pavol; Lebedev, Andrey A.; Pannu, Navraj S.; Steiner, Roberto A.; Nicholls, Robert A.; Winn, Martyn D.; Long, Fei; Vagin, Alexei A.
    Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 355-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    This paper describes various components of the macromol. crystallog. refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS exptl. diffraction data. To ensure chem. and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolns. at least as low as 4 Å can be achieved thanks to low-resoln. refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, 'jelly-body' restraints and the use of novel long-range restraints on at. displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 addnl. offers TLS parameterization and, when high-resoln. data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resoln. spectrum encountered in macromol. crystallog.
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    Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol Crystallogr 2004, 60, 2126– 2132,  DOI: 10.1107/S0907444904019158
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    Coot: model-building tools for molecular graphics
    Emsley, Paul; Cowtan, Kevin
    Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
    CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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    Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol Crystallogr 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
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    Features and development of Coot
    Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
    Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
    Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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    Langer, G. G.; Cohen, S. X.; Lamzin, V. S.; Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 2008, 3, 1171– 1179,  DOI: 10.1038/nprot.2008.91
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    Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7
    Langer, Gerrit; Cohen, Serge X.; Lamzin, Victor S.; Perrakis, Anastassis
    Nature Protocols (2008), 3 (7), 1171-1179CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    ARP/wARP is a software suite to build macromol. models in x-ray crystallog. electron d. maps. Structural genomics initiatives and the study of complex macromol. assemblies and membrane proteins all rely on advanced methods for 3D structure detn. ARP/wARP meets these needs by providing the tools to obtain a macromol. model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water mols. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.
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    Current Opinion in Structural Biology (1998), 8 (5), 631-639CODEN: COSBEF; ISSN:0959-440X. (Current Biology Publications)
    A review with 55 refs. The growing no. of protein structures solved at at. resoln. holds the promise of further improvements in geometry-based validation parameters. Addnl., the estd. std. uncertainties of the at. coordinates have been computed for a no. of x-ray structures, providing a measure of the coordinate precision. In NMR spectroscopy, a measure analogous to the crystallog. R-factor has been developed.
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    Potterton, L.; McNicholas, S.; Krissinel, E.; Gruber, J.; Cowtan, K.; Emsley, P.; Murshudov, G. N.; Cohen, S.; Perrakis, A.; Noble, M. Developments in the CCP4 molecular-graphics project. Acta Crystallogr. D Biol Crystallogr 2004, 60, 2288– 2294,  DOI: 10.1107/S0907444904023716
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    Developments in the CCP4 molecular-graphics project
    Potterton, Liz; McNicholas, Stuart; Krissinel, Eugene; Gruber, Jan; Cowtan, Kevin; Emsley, Paul; Murshudov, Garib N.; Cohen, Serge; Perrakis, Anastassis; Noble, Martin
    Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2288-2294CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
    Progress towards structure detn. that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-d. map interpretation and for the anal. of the resulting at. models. Advances in map-interpretation algorithms are extending the resoln. regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromol. electron d., particularly where crystallog. data is only available to modest resoln. [for example, I/σ(I) < 2.0 for min. resoln. 2.5 Å]. In such cases, a set of manual and semi-manual model-building mol.-graphics tools is needed. At the same time, converting the knowledge encapsulated in a mol. structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by both static and dynamic representations. CCP4mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biol. As well as providing a carefully designed user interface to advanced algorithms of model building and anal., CCP4mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.
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    Shanks, E. J.; Ong, H. B.; Robinson, D. A.; Thompson, S.; Sienkiewicz, N.; Fairlamb, A. H.; Frearson, J. A. Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase. Anal. Biochem. 2010, 396, 194– 203,  DOI: 10.1016/j.ab.2009.09.003
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    Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase
    Shanks, Emma J.; Ong, Han B.; Robinson, David A.; Thompson, Stephen; Sienkiewicz, Natasha; Fairlamb, Alan H.; Frearson, Julie A.
    Analytical Biochemistry (2010), 396 (2), 194-203CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidn. of NADP (NADPH). Although this assay has been adequate to study the biol. of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymically coupled screening assay in which the product of the enzymic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-mol. inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
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    Ferrari, S.; Morandi, F.; Motiejunas, D.; Nerini, E.; Henrich, S.; Luciani, R.; Venturelli, A.; Lazzari, S.; Calò, S.; Gupta, S.; Hannaert, V.; Michels, P. A. M.; Wade, R. C.; Costi, M. P. Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase. J. Med. Chem. 2011, 54, 211– 221,  DOI: 10.1021/jm1010572
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    Virtual Screening Identification of Nonfolate Compounds, Including a CNS Drug, as Antiparasitic Agents Inhibiting Pteridine Reductase
    Ferrari, Stefania; Morandi, Federica; Motiejunas, Domantas; Nerini, Erika; Henrich, Stefan; Luciani, Rosaria; Venturelli, Alberto; Lazzari, Sandra; Calo, Samuele; Gupta, Shreedhara; Hannaert, Veronique; Michels, Paul A. M.; Wade, Rebecca C.; Costi, M. Paola
    Journal of Medicinal Chemistry (2011), 54 (1), 211-221CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Folate analog inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new mols. with specificity for PTR1, we carried out a virtual screening of the Available Chems. Directory (ACD) database to select compds. that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like mols. with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compds. both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compds. showed efficacy only in combination. In toxicity tests against human fibroblasts, several compds. showed low toxicity. One compd., 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclin. evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.
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    The importance of loop region residues 40-46 in human dihydrofolate reductase as revealed by site-directed mutagenesis
    Tan, Xuehai; Huang, Shaoming; Ratnam, Manohar; Thompson, Paul D.; Freisheim, James H.
    Journal of Biological Chemistry (1990), 265 (14), 8027-32CODEN: JBCHA3; ISSN:0021-9258.
    Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible loop region between residues 40 and 46 of human dihydrofolate reductase. Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and NADPH as well as the pH rate profile are virtually identical to that of the wild type. In contrast, the Vmax of the mutant enzyme is decreased 2.5-fold. The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates. Agents such as KCl, urea, and organomercurials at concns. which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant. Competitive ELISA expts. using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking redn. in the binding of the antibodies. Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies. The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus communicating the effects of ligand binding.
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    Linciano, P.; Dawson, A.; Pöhner, I.; Costa, D. M.; Sá, M. S.; Cordeiro-da-Silva, A.; Luciani, R.; Gul, S.; Witt, G.; Ellinger, B.; Kuzikov, M.; Gribbon, P.; Reinshagen, J.; Wolf, M.; Behrens, B.; Hannaert, V.; Michels, P. A. M.; Nerini, E.; Pozzi, C.; Di Pisa, F.; Landi, G.; Santarem, N.; Ferrari, S.; Saxena, P.; Lazzari, S.; Cannazza, G.; Freitas-Junior, L. H.; Moraes, C. B.; Pascoalino, B. S.; Alcântara, L. M.; Bertolacini, C. P.; Fontana, V.; Wittig, U.; Müller, W.; Wade, R. C.; Hunter, W. N.; Mangani, S.; Costantino, L.; Costi, M. P. Exploiting the 2-Amino-1,3,4-thiadiazole scaffold to inhibit Trypanosoma brucei pteridine reductase in support of early-stage drug discovery. ACS Omega 2017, 2, 5666– 5683,  DOI: 10.1021/acsomega.7b00473
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    Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery
    Linciano, Pasquale; Dawson, Alice; Pohner, Ina; Costa, David M.; Sa, Monica S.; Cordeiro-da-Silva, Anabela; Luciani, Rosaria; Gul, Sheraz; Witt, Gesa; Ellinger, Bernhard; Kuzikov, Maria; Gribbon, Philip; Reinshagen, Jeanette; Wolf, Markus; Behrens, Birte; Hannaert, Veronique; Michels, Paul A. M.; Nerini, Erika; Pozzi, Cecilia; di Pisa, Flavio; Landi, Giacomo; Santarem, Nuno; Ferrari, Stefania; Saxena, Puneet; Lazzari, Sandra; Cannazza, Giuseppe; Freitas-Junior, Lucio H.; Moraes, Carolina B.; Pascoalino, Bruno S.; Alcantara, Laura M.; Bertolacini, Claudia P.; Fontana, Vanessa; Wittig, Ulrike; Muller, Wolfgang; Wade, Rebecca C.; Hunter, William N.; Mangani, Stefano; Costantino, Luca; Costi, Maria P.
    ACS Omega (2017), 2 (9), 5666-5683CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. The authors investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). The authors detd. crystal structures of several TbPTR1-inhibitor complexes to guide structure-based design of new thiadiazole derivs. Subsequent synthesis, enzyme and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compd. I, a biphenyl-thiadiazole-2,5-diamine, with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase (DHFR) inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. The antiparasitic activity of the (I + MTX) combination was reversed by addn. of folic acid. By adopting an efficient early hit discovery platform, the authors demonstrate with the 2-amino-1,3,4-thiadiazole scaffold how a promising tool for the development of anti-Trypanosoma brucei agents can be obtained.
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    Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.; Timlin, M. R.; Uchimaya, M. Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J. Comput. Aided Mol. Des. 2007, 21, 681– 691,  DOI: 10.1007/s10822-007-9133-z
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    Epik: a software program for pKa prediction and protonation state generation for drug-like molecules
    Shelley, John C.; Cholleti, Anuradha; Frye, Leah L.; Greenwood, Jeremy R.; Timlin, Mathew R.; Uchimaya, Makoto
    Journal of Computer-Aided Molecular Design (2007), 21 (12), 681-691CODEN: JCADEQ; ISSN:0920-654X. (Springer)
    Epik is a computer program for predicting pKa values for drug-like mols. Epik can use this capability in combination with technol. for tautomerization to adjust the protonation state of small drug-like mols. to automatically generate one or more of the most probable forms for use in further mol. modeling studies. Many medicinal chems. can exchange protons with their environment, resulting in various ionization and tautomeric states, collectively known as protonation states. The protonation state of a drug can affect its soly. and membrane permeability. In modeling, the protonation state of a ligand will also affect which conformations are predicted for the mol., as well as predictions for binding modes and ligand affinities based upon protein-ligand interactions. Despite the importance of the protonation state, many databases of candidate mols. used in drug development do not store reliable information on the most probable protonation states. Epik is sufficiently rapid and accurate to process large databases of drug-like mols. to provide this information. Several new technologies are employed. Extensions to the well-established Hammett and Taft approaches are used for pKa prediction, namely, mesomer standardization, charge cancellation, and charge spreading to make the predicted results reflect the nature of the mol. itself rather just for the particular Lewis structure used on input. In addn., a new iterative technol. for generating, ranking and culling the generated protonation states is employed.
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    Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J. Comput. Aided Mol. Des. 2010, 24, 591– 604,  DOI: 10.1007/s10822-010-9349-1
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    Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution
    Greenwood, Jeremy R.; Calkins, David; Sullivan, Arron P.; Shelley, John C.
    Journal of Computer-Aided Molecular Design (2010), 24 (6-7), 591-604CODEN: JCADEQ; ISSN:0920-654X. (Springer)
    A review. Generating the appropriate protonation states of drug-like mols. in soln. is important for success in both ligand- and structure-based virtual screening. Screening collections of millions of compds. requires a method for detg. tautomers and their energies that is sufficiently rapid, accurate, and comprehensive. To maximize enrichment, the lowest energy tautomers must be detd. from heterogeneous input, without over-enumerating unfavorable states. While computationally expensive, the d. functional theory (DFT) method M06-2X/aug-cc-pVTZ(-f) [PB-SCRF] provides accurate energies for enumerated model tautomeric systems. The empirical Hammett-Taft methodol. can very rapidly extrapolate substituent effects from model systems to drug-like mols. via the relationship between pKT and pKa. Combining the 2 complementary approaches transforms the tautomer problem from a scientific challenge to one of engineering scale-up, and avoids issues that arise due to the very limited no. of measured pKT values, esp. for the complicated heterocycles often favored by medicinal chemists for their novelty and versatility. Several hundreds of pre-calcd. tautomer energies and substituent pKa effects are tabulated in databases for use in structural adjustment by the program Epik, which treats tautomers as a subset of the larger problem of the protonation states in aq. ensembles and their energy penalties. Accuracy and coverage is continually improved and expanded by parameterizing new systems of interest using DFT and exptl. data. Recommendations are made for how to best incorporate tautomers in mol. design and virtual screening workflows.
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    Sanschagrin, P. C.; Kuhn, L. A. Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity. Protein Sci. 1998, 7, 2054– 2064,  DOI: 10.1002/pro.5560071002
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    Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity
    Sanschagrin, Paul C.; Kuhn, Leslie A.
    Protein Science (1998), 7 (10), 2054-2064CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
    Cluster anal. is presented as a technique for analyzing the conservation and chem. of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster anal. provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water mols., and also avoids reliance on any one structure as a ref. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water mols. from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concd. in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is assocd. with ligand selectivity. Cluster anal. also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addn. to facilitating the anal. of water sites from multiple structures, cluster anal. provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.
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    Madhavi Sastry, G.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 2013, 27, 221– 234,  DOI: 10.1007/s10822-013-9644-8
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    Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments
    Madhavi Sastry, G.; Adzhigirey, Matvey; Day, Tyler; Annabhimoju, Ramakrishna; Sherman, Woody
    Journal of Computer-Aided Molecular Design (2013), 27 (3), 221-234CODEN: JCADEQ; ISSN:0920-654X. (Springer)
    Structure-based virtual screening plays an important role in drug discovery and complements other screening approaches. In general, protein crystal structures are prepd. prior to docking in order to add hydrogen atoms, optimize hydrogen bonds, remove at. clashes, and perform other operations that are not part of the x-ray crystal structure refinement process. In addn., ligands must be prepd. to create 3-dimensional geometries, assign proper bond orders, and generate accessible tautomer and ionization states prior to virtual screening. While the prerequisite for proper system prepn. is generally accepted in the field, an extensive study of the prepn. steps and their effect on virtual screening enrichments has not been performed. In this work, we systematically explore each of the steps involved in prepg. a system for virtual screening. We first explore a large no. of parameters using the Glide validation set of 36 crystal structures and 1,000 decoys. We then apply a subset of protocols to the DUD database. We show that database enrichment is improved with proper prepn. and that neglecting certain steps of the prepn. process produces a systematic degrdn. in enrichments, which can be large for some targets. We provide examples illustrating the structural changes introduced by the prepn. that impact database enrichment. While the work presented here was performed with the Protein Prepn. Wizard and Glide, the insights and guidance are expected to be generalizable to structure-based virtual screening with other docking methods.
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    Li, H.; Robertson, A. D.; Jensen, J. H. Very fast empirical prediction and rationalization of protein pKa values. Proteins 2005, 61, 704– 721,  DOI: 10.1002/prot.20660
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    Very fast empirical prediction and rationalization of protein pKa values
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    Proteins: Structure, Function, and Bioinformatics (2005), 61 (4), 704-721CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    A very fast empirical method is presented for structure-based protein pKa prediction and rationalization. The desolvation effects and intra-protein interactions, which cause variations in pKa values of protein ionizable groups, are empirically related to the positions and chem. nature of the groups proximate to the pKa sites. A computer program is written to automatically predict pKa values based on these empirical relationships within a couple of seconds. Unusual pKa values at buried active sites, which are among the most interesting protein pKa values, are predicted very well with the empirical method. A test on 233 carboxyl, 12 cysteine, 45 histidine, and 24 lysine pKa values in various proteins shows a root-mean-square deviation (RMSD) of 0.89 from exptl. values. Removal of the 29 pKa values that are upper or lower limits results in an RMSD = 0.79 for the remaining 285 pKa values.
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    Banks, J. L.; Beard, H. S.; Cao, Y.; Cho, A. E.; Damm, W.; Farid, R.; Felts, A. K.; Halgren, T. A.; Mainz, D. T.; Maple, J. R.; Murphy, R.; Philipp, D. M.; Repasky, M. P.; Zhang, L. Y.; Berne, B. J.; Friesner, R. A.; Gallicchio, E.; Levy, R. M. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J. Comput. Chem. 2005, 26, 1752– 1780,  DOI: 10.1002/jcc.20292
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    Integrated modeling program, applied chemical theory (IMPACT)
    Banks, Jay L.; Beard, Hege S.; Cao, Yixiang; Cho, Art E.; Damm, Wolfgang; Farid, Ramy; Felts, Anthony K.; Halgren, Thomas A.; Mainz, Daniel T.; Maple, Jon R.; Murphy, Robert; Philipp, Dean M.; Repasky, Matthew P.; Zhang, Linda Y.; Berne, Bruce J.; Friesner, Richard A.; Gallicchio, Emilio; Levy, Ronald M.
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    A review. We provide an overview of the IMPACT mol. mechanics program with an emphasis on recent developments and a description of its current functionality. With respect to core mol. mechanics technologies we include a status report for the fixed charge and polarizable force fields that can be used with the program and illustrate how the force fields, when used together with new atom typing and parameter assignment modules, have greatly expanded the coverage of org. compds. and medicinally relevant ligands. As we discuss in this review, explicit solvent simulations have been used to guide our design of implicit solvent models based on the generalized Born framework and a novel nonpolar estimator that have recently been incorporated into the program. With IMPACT it is possible to use several different advanced conformational sampling algorithms based on combining features of mol. dynamics and Monte Carlo simulations. The program includes two specialized mol. mechanics modules: Glide, a high-throughput docking program, and QSite, a mixed quantum mechanics/mol. mechanics module. These modules employ the IMPACT infrastructure as a starting point for the construction of the protein model and assignment of mol. mechanics parameters, but have then been developed to meet specialized objectives with respect to sampling and the energy function.
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    Halgren, T. New method for fast and accurate binding-site identification and analysis. Chem. Biol. Drug Des. 2007, 69, 146– 148,  DOI: 10.1111/j.1747-0285.2007.00483.x
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    New method for fast and accurate binding-site identification and analysis
    Halgren, Tom
    Chemical Biology & Drug Design (2007), 69 (2), 146-148CODEN: CBDDAL; ISSN:1747-0277. (Blackwell Publishing Ltd.)
    Structure-based drug design seeks to exploit the structure of protein-ligand or protein-protein binding sites, but the site is not always known at the outset. Even when the site is known, the researcher may wish to identify alternative prospective binding sites that may result in different biol. effects or new class of compds. It is also vital in lead optimization to clearly understand the degree to which known binders or docking hits satisfy or violate complementarity to the receptor. SiteMap is a new technique for identifying potential binding sites and for predicting their druggability in lead-discovery applications and for characterizing binding sites and critically assessing prospective ligands in lead-optimization applications. In large-scale validation tests, SiteMap correctly identifies the known binding site in > 96% of the cases, with best results (> 98%) coming for sites that bind ligands tightly. It also accurately distinguishes between sites that bind ligands and sites that don't. In binding-site anal., SiteMap provides a wealth of quant. and graphical information that can help guide efforts to modify ligand structure to enhance potency or improve phys. properties. These attributes allow SiteMap to nicely complement techniques such as docking and computational lead optimization in structure-base drug design.
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    Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 2009, 49, 377– 389,  DOI: 10.1021/ci800324m
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    Identifying and Characterizing Binding Sites and Assessing Druggability
    Halgren, Thomas A.
    Journal of Chemical Information and Modeling (2009), 49 (2), 377-389CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    Identification and characterization of binding sites is key in the process of structure-based drug design. In some cases there may not be any information about the binding site for a target of interest. In other cases, a putative binding site has been identified by computational or exptl. means, but the druggability of the target is not known. Even when a site for a given target is known, it may be desirable to find addnl. sites whose targeting could produce a desired biol. response. A new program, called SiteMap, is presented for identifying and analyzing binding sites and for predicting target druggability. In a large-scale validation, SiteMap correctly identifies the known binding site as the top-ranked site in 86% of the cases, with best results (>98%) coming for sites that bind ligands with subnanomolar affinity. In addn., a modified version of the score employed for binding-site identification allows SiteMap to accurately classify the druggability of proteins as measured by their ability to bind passively absorbed small mols. tightly. In characterizing binding sites, SiteMap provides quant. and graphical information that can help guide efforts to critically assess virtual hits in a lead-discovery application or to modify ligand structure to enhance potency or improve phys. properties in a lead-optimization context.
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    Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739– 1749,  DOI: 10.1021/jm0306430
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    Glide: A new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy
    Friesner, Richard A.; Banks, Jay L.; Murphy, Robert B.; Halgren, Thomas A.; Klicic, Jasna J.; Mainz, Daniel T.; Repasky, Matthew P.; Knoll, Eric H.; Shelley, Mee; Perry, Jason K.; Shaw, David E.; Francis, Perry; Shenkin, Peter S.
    Journal of Medicinal Chemistry (2004), 47 (7), 1739-1749CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Unlike other methods for docking ligands to the rigid 3D structure of a known protein receptor, Glide approximates a complete systematic search of the conformational, orientational, and positional space of the docked ligand. In this search, an initial rough positioning and scoring phase that dramatically narrows the search space is followed by torsionally flexible energy optimization on an OPLS-AA nonbonded potential grid for a few hundred surviving candidate poses. The very best candidates are further refined via a Monte Carlo sampling of pose conformation; in some cases, this is crucial to obtaining an accurate docked pose. Selection of the best docked pose uses a model energy function that combines empirical and force-field-based terms. Docking accuracy is assessed by redocking ligands from 282 cocrystd. PDB complexes starting from conformationally optimized ligand geometries that bear no memory of the correctly docked pose. Errors in geometry for the top-ranked pose are less than 1 Å in nearly half of the cases and are greater than 2 Å in only about one-third of them. Comparisons to published data on rms deviations show that Glide is nearly twice as accurate as GOLD and more than twice as accurate as FlexX for ligands having up to 20 rotatable bonds. Glide is also found to be more accurate than the recently described Surflex method.
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    Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 2004, 47, 1750– 1759,  DOI: 10.1021/jm030644s
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    Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening
    Halgren, Thomas A.; Murphy, Robert B.; Friesner, Richard A.; Beard, Hege S.; Frye, Leah L.; Pollard, W. Thomas; Banks, Jay L.
    Journal of Medicinal Chemistry (2004), 47 (7), 1750-1759CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Glide's ability to identify active compds. in a database screen is characterized by applying Glide to a diverse set of nine protein receptors. In many cases, two, or even three, protein sites are employed to probe the sensitivity of the results to the site geometry. To make the database screens as realistic as possible, the screens use sets of "druglike" decoy ligands that have been selected to be representative of what we believe is likely to be found in the compd. collection of a pharmaceutical or biotechnol. company. Results are presented for releases 1.8, 2.0, and 2.5 of Glide. The comparisons show that av. measures for both "early" and "global" enrichment for Glide 2.5 are 3 times higher than for Glide 1.8 and more than 2 times higher than for Glide 2.0 because of better results for the least well-handled screens. This improvement in enrichment stems largely from the better balance of the more widely parametrized GlideScore 2.5 function and the inclusion of terms that penalize ligand-protein interactions that violate established principles of phys. chem., particularly as it concerns the exposure to solvent of charged protein and ligand groups. Comparisons to results for the thymidine kinase and estrogen receptors published by Rognan and co-workers (J. Med. Chem. 2000, 43, 4759-4767) show that Glide 2.5 performs better than GOLD 1.1, FlexX 1.8, or DOCK 4.01.
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    Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra Precision Glide: Docking and scoring incorporating a model of hydrophobic wnclosure for protein-ligand complexes. J. Med. Chem. 2006, 49, 6177– 6196,  DOI: 10.1021/jm051256o
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    Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes
    Friesner, Richard A.; Murphy, Robert B.; Repasky, Matthew P.; Frye, Leah L.; Greenwood, Jeremy R.; Halgren, Thomas A.; Sanschagrin, Paul C.; Mainz, Daniel T.
    Journal of Medicinal Chemistry (2006), 49 (21), 6177-6196CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A novel scoring function to est. protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addn. to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included:(1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce exptl. binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, resp.) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP mol. recognition and water scoring in sepg. active and inactive ligands and avoiding false positives.
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    Sherman, W.; Beard, H. S.; Farid, R. Use of an induced fit receptor structure in virtual screening. Chem. Biol. Drug Des. 2006, 67, 83– 84,  DOI: 10.1111/j.1747-0285.2005.00327.x
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    Use of an induced fit receptor structure in virtual screening
    Sherman, Woody; Beard, Hege S.; Farid, Ramy
    Chemical Biology & Drug Design (2006), 67 (1), 83-84CODEN: CBDDAL; ISSN:1747-0277. (Blackwell Publishing Ltd.)
    A review. The automated induced fit docking protocol was used to generate the DFG-out conformation from a p38 MAP kinase activation loop starting from a DFG-in structure (1a9u) and the ligand from 1kv1 (BMU). In a virtual screening study of 25K decoy ligands and 46 known actives, using an ensemble consisting of the induced fit docking structure (DFG-out) and the 1a9u crystal structure (DFG-in), 14 actives were identified in the top 1% of the database, including BMU and BIRB 796. 3 Actives were identified when 1a9u was used alone.
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    Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. Novel procedure for modeling ligand/receptor induced fit effects. J. Med. Chem. 2006, 49, 534– 553,  DOI: 10.1021/jm050540c
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    Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects
    Sherman, Woody; Day, Tyler; Jacobson, Matthew P.; Friesner, Richard A.; Farid, Ramy
    Journal of Medicinal Chemistry (2006), 49 (2), 534-553CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We present a novel protein-ligand docking method that accurately accounts for both ligand and receptor flexibility by iteratively combining rigid receptor docking (Glide) with protein structure prediction (Prime) techniques. While traditional rigid-receptor docking methods are useful when the receptor structure does not change substantially upon ligand binding, success is limited when the protein must be "induced" into the correct binding conformation for a given ligand. We provide an in-depth description of our novel methodol. and present results for 21 pharmaceutically relevant examples. Traditional rigid-receptor docking for these 21 cases yields an av. RMSD of 5.5 Å. The av. ligand RMSD for docking to a flexible receptor for the 21 pairs is 1.4 Å; the RMSD is ≤1.8 Å for 18 of the cases. For the three cases with RMSDs greater than 1.8 Å, the core of the ligand is properly docked and all key protein/ligand interactions are captured.
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    The application of all-atom force fields (and explicit or implicit solvent models) to protein homol.-modeling tasks such as side-chain and loop prediction remains challenging both because of the expense of the individual energy calcns. and because of the difficulty of sampling the rugged all-atom energy surface. Here the authors address this challenge for the problem of loop prediction through the development of numerous new algorithms, with an emphasis on multiscale and hierarchical techniques. As a first step in evaluating the performance of the authors' loop prediction algorithm, the authors have applied it to the problem of reconstructing loops in native structures; the authors also explicitly include crystal packing to provide a fair comparison with crystal structures. In brief, large nos. of loops are generated by using a dihedral angle-based buildup procedure followed by iterative cycles of clustering, side-chain optimization, and complete energy minimization of selected loop structures. The authors evaluate this method by the largest test set yet used for validation of a loop prediction method, with a total of 833 loops ranging from 4 to 12 residues in length. Av./median backbone root-mean-square deviations (RMSDs) to the native structures (superimposing the body of the protein, not the loop itself) are 0.42/0.24 Å for 5 residue loops, 1.00/0.44 Å for 8 residue loops, and 2.47/1.83 Å for 11 residue loops. Median RMSDs are substantially lower than the avs. because of a small no. of outliers; the causes of these failures are examd. in some detail, and many can be attributed to errors in assignment of protonation states of titratable residues, omission of ligands from the simulation, and, in a few cases, probable errors in the exptl. detd. structures. When these obvious problems in the data sets are filtered out, av. RMSDs to the native structures improve to 0.43 Å for 5 residue loops, 0.84 Å for 8 residue loops, and 1.63 Å for 11 residue loops. In the vast majority of cases, the method locates energy min. that are lower than or equal to that of the minimized native loop, thus indicating that sampling rarely limits prediction accuracy. The overall results are, to the authors' knowledge, the best reported to date, and the authors attribute this success to the combination of an accurate all-atom energy function, efficient methods for loop buildup and side-chain optimization, and, esp. for the longer loops, the hierarchical refinement protocol.
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    Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei, and the disease is fatal if untreated. There is an urgent need to develop new, safe and effective treatments for HAT because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the development and application of a cell-based resazurin reduction assay for high throughput screening and identification of new inhibitors of T. b. brucei as starting points for the development of new treatments for human HAT. Active compounds identified in primary screening of ∼48,000 compounds representing ∼25 chemical classes were titrated to obtain IC50 values. Cytotoxicity against a mammalian cell line was determined to provide indications of parasite versus host cell selectivity. Examples from hit series that showed selectivity and evidence of preliminary SAR were re-synthesized to confirm trypanocidal activity prior to initiating hit-to-lead expansion efforts. Additional assays such as serum shift, time to kill and reversibility of compound effect were developed and applied to provide further criteria for advancing compounds through the hit-to-lead phase of the project. From this initial effort, six distinct chemical series were selected and hit-to-lead chemistry was initiated to synthesize several key analogs for evaluation of trypanocidal activity in the resazurin-reduction assay for parasite viability. From the hit-to-lead efforts, a series was identified that demonstrated efficacy in a mouse model for T. b. brucei infection and was progressed into the lead optimization stage. In summary, the present study demonstrates the successful and effective use of resazurin-reduction based assays as tools for primary and secondary screening of a new compound series to identify leads for the treatment of HAT.
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    The LIBRA compd. library is a collection of 522 non-com. mols. contributed by various Italian academic labs. These compds. have been designed and synthesized during different medicinal chem. programs and are hosted by the Italian Institute of Technol. We report the screening of the LIBRA compd. library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compds. were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivs. were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metab., excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivs.
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    Journal of Pharmacology and Experimental Therapeutics (2008), 327 (3), 918-925CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)
    The effects of feeding diets high and low in folic acid (folate) on assocd. changes in blood serum and red blood cell (RBC) folate levels, tissue-derived folate receptor levels, and the ability of folate-drug conjugates (EC-145, EC-0225, EC-0305) to bind and exert activity against folate receptor (FR)-pos. KB tumor xenografts were studied in lab. mice. Serum and RBC folate concns. sharply decreased immediately after the mice were switched to low folate diets, but both parameters reached steady-state ("human-like") levels after 6 wk. The tissue-related folate binding capacities were also decreased during the dietary modulation period, whereas the net uptake of radiolabeled folate conjugate was simultaneously increased 2.6- and 5-fold in FR-pos. kidney and tumor tissues, resp. The performances of several clin. and preclinically relevant folate-drug conjugates were evaluated against tumors in mice that were fed high or low folate diets. Except when administered at doses 6-fold less than required to sat. endogenous FR, no significant loss of antitumor activity was obsd. Thus, lowering the dietary intake of folates in mice had little impact on the biol. activity of repetitively dosed folate-targeted agents, but low folate diet regimens decreased blood serum and RBC folate levels down to levels that more closely approx. the normal human folate ranges.
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    The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 microg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC50s, of 0.54 and 1.32 microg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.
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Cited By

This article is cited by 2 publications.

  1. Joanna Panecka-Hofman, Ina Poehner, Rebecca C. Wade. Anti-trypanosomatid structure-based drug design – lessons learned from targeting the folate pathway. Expert Opinion on Drug Discovery 2022, 17 (9) , 1029-1045. https://doi.org/10.1080/17460441.2022.2113776
  2. Alberto Venturelli, Lorenzo Tagliazucchi, Clara Lima, Federica Venuti, Giulia Malpezzi, George E. Magoulas, Nuno Santarem, Theodora Calogeropoulou, Anabela Cordeiro-da-Silva, Maria Paola Costi. Current Treatments to Control African Trypanosomiasis and One Health Perspective. Microorganisms 2022, 10 (7) , 1298. https://doi.org/10.3390/microorganisms10071298
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  • Abstract

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    Figure 1

    Figure 1. Overview of pterin activation in the trypanosomatidic folate pathway when DHFR is inhibited and PTR1 provides a metabolic bypass. Under normal conditions (indicated by dashed lines), the DHFR domain of the bifunctional DHFR-TS reduces biological folates to tetrahydrofolate (THF). Serine hydroxymethyl transferase (SHMT) converts THF to 5,10-methylene THF, which has a central role in amino acid synthesis, protein biosynthesis, and one-carbon transfer. It is also required by the TS domain of DHFR-TS to convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), which is necessary for DNA synthesis. PTR1 catalyzes the reduction of unconjugated pterins, like biopterin, and takes over folate reduction when DHFR is inhibited (continuous lines), thus acting as a metabolic bypass and an important additional target for shutting down the trypanosomatidic folate pathway. Both proteins are shown in cartoon representation (DHFR domain of DHFR-TS: purple, PTR1 monomer of the functional tetramer: light pink) with the NADPH/NADP+ cofactor in a stick representation with black carbons and the folate substrate in yellow spheres. In PTR1, an arginine residue from a neighboring subunit that points into the active site is shown in a magenta stick representation.

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    Figure 2

    Figure 2. Inhibitory activities, selectivities, and structures of reference pteridines. (A) Heatmaps show activities given by IC50 values (top) and selectivity indices (SI) (bottom) for the targets and the off-target hDHFR. All values, as well as data for hTS, are given in Table S1. NI: no inhibition; NA: not applicable. (B) Previously published compounds shown were used as reference compounds: 1a is methotrexate; 1b, 1c, and 1h are 6b, 6a, and 6c from Cavazzuti et al.; (26) and 1d–1g correspond to 5d, 5b, 6a, and 5a from Corona et al. (30)

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    Figure 3

    Figure 3. Orientations of reference pteridine compound 1b in crystal structures of LmPTR1 and TbPTR1. (A,B) Compound 1b (cyan carbons) in complex with LmPTR1 (PDB-ID 2qhx) has a substrate-like (A) and an inhibitor-like or MTX-like (B) binding mode. 1b is shown with (A) folate (yellow carbons) superimposed from a TbPTR1 structure (PDB-ID 3bmc) and with (B) MTX (1a, yellow carbons) superimposed from an LmPTR1 structure (PDB-ID 1e7w). The pteridine nitrogens are labeled according to the ring nomenclature. (C) Binding site in the crystal structure determined in this work (PDB-ID 6rx5) of TbPTR1 (gray cartoon, His267′ from the neighboring subunit in lavender) in complex with NADPH/NADP+ and compound 1b, which has the MTX-like binding mode. Interacting residues (in A, B: only Phe113) and the NADPH/NADP+ cofactor are shown in sticks (carbons colored according to protein and black, respectively). In (C), water molecules are shown as red spheres, and the inhibitor is surrounded by the omit map (green wire) contoured at the 2.5 σ level. Hydrogen bonds are represented by brown dashed lines.

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    Figure 4

    Figure 4. Structural features of PTR1 and DHFR considered in the multitarget design of selective compounds illustrated for reference compound 1b. (A) Selected residues within 5 Å of the three modules─N10, PABA and Tail─modified in the design procedure. Residues were selected for the complexes of 1b with TbPTR1 (pale gray), TbDHFR (dark gray), LmPTR1 (pale pink), and LmDHFR (dark pink). Residues are colored according to their properties: basic: blue, polar: green, and nonpolar: yellow. The ligand interaction plot is based on Panecka-Hofman et al. (31) and provides an overview of residues with similar properties that surround the ligand modules in the different targets (showing only those applied for the design; for full maps, see Figures S3 and S4). In some positions, the amino acid type of the off-target hDHFR is different from parasite DHFR. Differing hDHFR residues are labeled in the top right corner of the corresponding parasite DHFR residue. These positions highlight suitable substitution points to improve selectivity. (B) Surface representations of complexes of 1b with TbPTR1 (left, PDB-ID 6rx5) and TbDHFR (right, MTX-like top-ranked docking pose in PDB-ID 3rg9). The compound tail moiety is fully solvent-exposed in PTR1, whereas it is well-enclosed in DHFR. (C) Surface representations of complexes of 1b with TbPTR1 (left, PDB-ID 6rx5) and LmPTR1 (right, PDB-ID 2qhx, state A). The ligand is more enclosed in the narrow pocket entrance of TbPTR1, while the LmPTR1 pocket has an elongated, widened funnel that can accommodate larger compound tails. In (B,C), 1b is shown in sticks with cyan carbons.

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    Figure 5

    Figure 5. Views of the binding sites showing docked poses of selected pteridine-based inhibitors in the target proteins: TbPTR1 (pale gray) (A,E), TbDHFR (dark gray) (B,F), LmPTR1 (pale pink) (C), and LmDHFR (dark pink) (D). (A) Induced fit (IF) MTX-like docking pose for compound 2c (cyan carbons) in TbPTR1 in the presence of a conserved water molecule (ball-and-stick representation): Trp221 moves (indicated by a brown arrow) to make room for the phenyl of 2c. (B–F) Rigid-body docking poses of 2c in TbDHFR (B), 3c (lime carbons) in LmPTR1 and LmDHFR (C,D), and 4e (purple carbons) in TbPTR1 and TbDHFR (E,F); see text for discussion. Docked poses are shown for N1-deprotonated compounds, but similar orientations were observed for the N1-protonated forms (see Figure S6). For PTR1, all docking poses shown were obtained in the presence of conserved structural water molecules. Generally, similar poses were observed for docking without water. In all panels, proteins are shown in cartoon representation with the important interacting residues (compare Figure 4A) and the NADPH/NADP+ cofactor shown in sticks (carbons colored according to protein and black, respectively). Residues His267′ and Arg287′ from the neighboring subunit are shown in lavender and magenta in TbPTR1 and LmPTR1, respectively. Hydrogen bonds are represented by brown dashed lines. Further IF docking poses are shown in Figures S7 and S8.

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    Figure 6

    Figure 6. Overview of the modifications in the N10, PABA, and Tail modules explored in the designed compound series with respect to the reference compound 1b. Synthesized members of each designed series are shown in the framed boxes along with the key objectives addressed with the respective modifications. See text for details.

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    Scheme 1

    Scheme 1. Synthesis of Derivatives of Compound 29a
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    aReagents and conditions: (i) SOCl2, reflux, 12 h, 70% yield; (ii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 60°C, 20′–30′ MW.

    Scheme 2

    Scheme 2. Synthesis of Compounds 1b,c, 2a–c, 2e, 4a–c, and 5c, and Intermediates 32–35, 37, 38, and 40–49a
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    aReagents and conditions: compounds 30, 31, 36, and 39 were purchased from Sigma; (i) acetonitrile or 3-hydroxypropanenitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24–36 h (32, 33); (ii) alkyl halide (propargyl bromide, (bromomethyl)benzene) (0.5 equiv), K2CO3 (2 equiv), DMF dry, rt, 24 h (34, 35); (iii) SOCl2 (4 equiv), propanol (for 37), EtOH (for 38), reflux, 7–12 h (89 and 96% yield); (iv) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (40–49); (v) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (1b,c, 2a–c, 2e, 4a–c, 5c).

    Scheme 3

    Scheme 3. Synthesis of Compounds 3a and 5a,ba
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    aReagents and conditions: (i) 3-hydroxypropanenitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24 h (51); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (52–54); (iii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (3a, 5a,b).

    Scheme 4

    Scheme 4. Synthesis of Compounds 4f,ga
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    aReagents and conditions: (i) di-tert-butyl pyrocarbonate (1.05 equiv), dioxane/H2O/1 N NaOH 1/1/1 V/V/V, rt, 6 h (55); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (56 and 57); (iii) TFA, DCM, rt (58 and 59); (iv) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (4f,g).

    Scheme 5

    Scheme 5. Synthesis of Compounds 4h–j and 5e,fa
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    aReagents and conditions: (i) K2CO3 (3 equiv), DMF, reflux, 16–18 h (63–65); (ii) NH2OH·HCl (1.2 equiv), EtOH, rt, >1 h followed by Zn dust (2.5 equiv) in 12 M HCl (4 equiv), rt, 15′ (66–68); (iii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (4h–j, 5e,f); (iv) methylamine (for 69) or benzylamine (for 70), EtOH dry, 60°C, 3 h, then NaBH4 (1.5 equiv), rt, 2 h.

    Scheme 6

    Scheme 6. Synthesis of Compounds 3b and 2da
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    aReagents and conditions: (i) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (71 and 72); (ii) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 20′ MW (3b, 2d).

    Scheme 7

    Scheme 7. Synthesis of Compounds 3c, 4d,e, and 5da
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    aReagents and conditions: (i) 29 (1.2 equiv), corresponding amine derivative (1 equiv), K2CO3 (3 equiv), KI (0.1 equiv), DMA, 30′ MW (3c, 4d,e, and 5d); (ii) acetonitrile, 10% Pd/C, NH4OAc (1 equiv), CH3OH, H2, rt, 24–36 h (74); (iii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight (75).

    Scheme 8

    Scheme 8. Synthesis of Compounds 3d,ea
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    aReagents and conditions: (i) KMnO4, acetone/0.5 M phosphate buffer at pH 7 (1:1 V/V); (ii) EDC·HCl (1.1 equiv), HOBt (0.1 equiv), TEA (2–3 equiv), DMF, rt, overnight.

    Figure 7

    Figure 7. Views of the binding sites of crystal structures of complexes of pteridine-based inhibitors in TbPTR1 and LmPTR1 determined in this work, which confirm the predicted MTX-like binding modes. (A) 2a (green carbons) in TbPTR1 (gray cartoon, His267′ from the neighboring subunit in lavender) and (B) 2e (yellow carbons) in LmPTR1 (pink cartoon, Arg287′ from the neighboring subunit in magenta). Water molecules are shown as red spheres, and the inhibitors are surrounded by the omit map (green wire) contoured at the 2.5 σ level. Interacting residues and the NADPH/NADP+ cofactor are shown in sticks (carbons colored according to protein and black, respectively). Hydrogen bonds are represented by brown dashed lines.

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    Figure 8

    Figure 8. Inhibitory activities (IC50 values, left) and selectivities (selectivity indices (SI), right) of compounds of the designed N10-, PABA-, and Tail-modified series and selected reference compounds against the targets TbPTR1, LmPTR1, TbDHFR, and LmDHFR and the off-target hDHFR. All values, as well as data for hTS, are reported in Table S1. Greener boxes show higher inhibition and selectivity. $ indicates that a precise activity value could not be determined as the tight binding limit was approached.

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    Figure 9

    Figure 9. Docking poses for compound 4c from the Tail series (magenta carbons) in (A,B) TbDHFR and (C) hDHFR, showing differences in exposure and interactions of the PABA and Tail moieties in the two DHFRs. (A) TbDHFR pocket accommodates 4c with its tail enclosed by surrounding residues. hDHFR has a similar shape. TbDHFR is shown in a gray surface representation. (B,C) Views of the binding sites of TbDHFR and hDHFR, which are shown in cartoon representation in gray and green, respectively. Important interacting residues and the NADPH/NADP+ cofactor (black carbons) are shown as sticks. Hydrogen bonds are indicated by brown dotted lines. While the orientations of 4c are rather similar in both DHFR variants, the tail moiety is more solvent-exposed in TbDHFR: the PABA benzene and piperidine of 4c compete for interactions with Phe94 of TbDHFR, which thereby becomes exposed to the solvent. In hDHFR, the corresponding exposed residue is the polar Asn64, and the tail of 4c can interact with Phe31 deeper in the pocket, rendering the mode of binding more favorable in hDHFR. The results are presented for N1-deprotonated compounds, but similar observations were made with N1-protonated compounds (Figure S6).

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    Figure 10

    Figure 10. Antiparasitic activity expressed as percentage of inhibition against T. brucei brucei for reference compounds and members of the N10-, PABA-, and Tail-modified series (A) and the selected representatives of the merged in silico library (B). The average of at least three independent determinations is shown with the standard deviation. The inactive compounds in the Tail-modified series, 4f, 4h, and 4j were omitted. Activities can be found in Table S7.

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    Figure 11

    Figure 11. Inhibitory activities, selectivities, and structures of the merged series of six pteridine derivatives. (A) Activity heatmap in the top panel shows IC50 values for the targets TbPTR1, LmPTR1, TbDHFR, and LmDHFR and the off-target hDHFR. All values, as well as data for hTS, are reported in Table S1. $ indicates that a precise activity value could not be determined as the tight binding limit was approached. In the bottom panel, selectivity indices are reported. (B) Structures of the selected and synthesized pteridines in the merged series.

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    Figure 12

    Figure 12. Heatmap representation of the liability assessment results for all the compounds studied. Inhibition of hERG as well as five CYP isoforms (1A2, 2C9, 2C19, 2D6, and 3A4), mitochondrial toxicity (MITO), and growth inhibition of A549 cells were determined at 10 μM. The data are represented as percentages on a color scale from white (desired) to orange (undesired) with values reported in the map. For the inhibitory activities against hERG, CYP isoforms, and mitochondrial toxicity, white = 0% and orange = 100% inhibition/toxicity, while for A549 cell growth inhibition, white = 100% and orange = 0% growth. The values are reported in Tables S8 and S9.

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      Expert Opinion on Therapeutic Patents (2014), 24 (6), 687-697CODEN: EOTPEG; ISSN:1354-3776. (Informa Healthcare)
      A review. The folate biosynthetic pathway, responsible for the de novo synthesis of thymidine and other key cellular components, is essential in all life forms and is esp. crit. in rapidly proliferating cells. As such, druggable targets along this pathway offer opportunities to impact many disease states such as cancer, infectious disease and autoimmune disease. In this article, recent progress on the development of antifolate compds. is reviewed. The evaluation of the patent literature during the period 2010 - 2013 focused on any compds. inhibiting recognized targets on the folate biosynthetic pathway. The folate pathway constitutes a well-validated and well-characterized set of targets; this pathway continues to elicit considerable enthusiasm for new drug discovery from both academic and industrial pharmaceutical research groups. Within the pathway, the enzymes dihydrofolate reductase and thymidylate synthase persist as the most attractive targets for new drug discovery for the treatment of cancer and infectious disease. Importantly, new potential targets for antifolates such as those on the purine biosynthetic pathway have been recently explored. The use of structure-based drug design is a major aspect in modern approaches to these drug targets.
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    15. 15
      Hawser, S.; Lociuro, S.; Islam, K. Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol. 2006, 71, 941– 948,  DOI: 10.1016/j.bcp.2005.10.052
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      Dihydrofolate reductase inhibitors as antibacterial agents
      Hawser, Stephen; Lociuro, Sergio; Islam, Khalid
      Biochemical Pharmacology (2006), 71 (7), 941-948CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)
      A review. Although only a few DHFR inhibitors have progressed as antibiotics to the market there is much renewed interest in the discovery and development of new generation DHFR inhibitors as antibacterial agents. This article describes the success in exploiting DHFR as a drugable target as exemplified by trimethoprim (TMP) and the development of several new diaminopyrimidines. Iclaprim, a recent example of a novel diaminopyrimidine currently in Phase III clin. trials, is also described together with several examples of anti-DHFR antibacterial compds. in pre-clin. development.
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    16. 16
      Yuthavong, Y.; Yuvaniyama, J.; Chitnumsub, P.; Vanichtanankul, J.; Chusacultanachai, S.; Tarnchompoo, B.; Vilaivan, T.; Kamchonwongpaisan, S. Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors. Parasitology 2005, 130, 249– 259,  DOI: 10.1017/S003118200400664X
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      16
      Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors
      Yuthavong, Y.; Yuvaniyama, J.; Chitnumsub, P.; Vanichtanankul, J.; Chusacultanachai, S.; Tarnchompoo, B.; Vilaivan, T.; Kamchonwongpaisan, S.
      Parasitology (2005), 130 (3), 249-259CODEN: PARAAE; ISSN:0031-1820. (Cambridge University Press)
      A review. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific addnl. sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes assocd. with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent antimalarial activities against drug-resistant P. falciparum bearing the mutant enzymes.
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      Christensen, K. E.; MacKenzie, R. E. Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals. Bioessays 2006, 28, 595– 605,  DOI: 10.1002/bies.20420
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      17
      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals
      Christensen, Karen E.; MacKenzie, Robert E.
      BioEssays (2006), 28 (6), 595-605CODEN: BIOEEJ; ISSN:0265-9247. (John Wiley & Sons, Inc.)
      A review. In eukaryotes, folate metab. is compartmentalized between the cytoplasm and organelles. The folate pathways of mitochondria are adapted to serve the metab. of the organism. In yeast, mitochondria support cytoplasmic purine synthesis through the generation of formate. This pathway is important but not essential for survival, consistent with the flexibility of yeast metab. In plants, the mitochondrial pathways support photorespiration by generating serine from glycine. This pathway is essential under photosynthetic conditions and the enzyme expression varies with photosynthetic activity. In mammals, the expression of the mitochondrial enzymes varies in tissues and during development. In embryos, mitochondria supply formate and glycine for purine synthesis, a process essential for survival; in adult tissues, flux through mitochondria can favor serine prodn. Thus, the differences in the folate pathways of mitochondria depending on species, tissues and developmental stages, profoundly alter the nature of their metabolic contribution.
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    18. 18
      Cullia, G.; Tamborini, L.; Conti, P.; De Micheli, C.; Pinto, A. Folates in Trypanosoma brucei: Achievements and opportunities. ChemMedChem. 2018, 13, 2150– 2158,  DOI: 10.1002/cmdc.201800500
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      18
      Folates in Trypanosoma brucei: Achievements and Opportunities
      Cullia, Gregorio; Tamborini, Lucia; Conti, Paola; De Micheli, Carlo; Pinto, Andrea
      ChemMedChem (2018), 13 (20), 2150-2158CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Trypanosoma brucei is the agent of human African trypanosomiasis (HAT), a neglected disease that threatens the lives of 65 million people in sub-Saharan Africa every year. Unfortunately, available therapies are unsatisfactory, due primarily to safety issues and development of drug resistance. Over the last decades significant effort has been made in the discovery of new potential anti-HAT agents, with help from the World Health Organization (WHO) and private-public partnerships such as the Drugs for Neglected Diseases Initiative (DNDi). Whereas antifolates have been a valuable source of drugs against bacterial infections and malaria, compds. effective against T. brucei have not yet been identified. Considering the relatively simple folate metabolic pathway in T. brucei, along with results obtained in this research field so far, we believe that further investigations might lead to effective chemotherapeutic agents. Herein we present a selection of the more promising results obtained so far in this field, underlining the opportunities that could lead to successful therapeutic approaches in the future.
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    19. 19
      Bello, A. R.; Nare, B.; Freedman, D.; Hardy, L.; Beverley, S. M. PTR1: A reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11442– 11446,  DOI: 10.1073/pnas.91.24.11442
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      19
      PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major
      Bello, Alexandre R.; Nare, Bakela; Freedman, Daniel; Hardy, Larry; Beverley, Stephen M.
      Proceedings of the National Academy of Sciences of the United States of America (1994), 91 (24), 11442-6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Trypanosomatid protozoans are pterin auxotrophs, a finding noted decades ago which heralded the discovery of key metabolic roles played by pteridines in eukaryotes. We have now identified the enzyme mediating unconjugated pteridine salvage in the human parasite Leishmania major. PTR1 is the gene in the amplified H region responsible for methotrexate (MTX) resistance, and belongs to a large family of oxidoreductases with diverse substrates and roles. We generated Leishmania lacking PTR1 by homologous gene targeting, and these ptr1- mutants required reduced biopterin (dihydro- or tetrahydrobiopterin) for growth. PTR1 purified from engineered Escherichia coli exhibited a MTX-sensitive, NADPH-dependent biopterin reductase activity. PTR1 showed good activity with folate and significant activity with dihydrofolate and dihydrobiopterin, but not with quinonoid dihydrobiopterin. PTR1 thus differs considerably from previously reported pteridine reductases of trypanosomatids and vertebrates. Pteridine reductase activity was diminished in ptr1- Leishmania and was evaluated in transfected parasites bearing multiple copies of PTR1; correspondingly, ptr1- was MTX-hypersensitive whereas the multicopy transfectant was MTX-resistant. The concordance of the biochem. and genetic properties of PTR1 suggests that this is the primary enzyme mediating pteridine salvage. These findings suggest several possible mechanisms for PTR1-mediated MTX resistance and should aid in the design of rational chemotherapy.
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    20. 20
      Dawson, A.; Gibellini, F.; Sienkiewicz, N.; Tulloch, L. B.; Fyfe, P. K.; McLuskey, K.; Fairlamb, A. H.; Hunter, W. N. Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate. Mol. Microbiol. 2006, 61, 1457– 1468,  DOI: 10.1111/j.1365-2958.2006.05332.x
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      20
      Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate
      Dawson, Alice; Gibellini, Federica; Sienkiewicz, Natasha; Tulloch, Lindsay B.; Fyfe, Paul K.; McLuskey, Karen; Fairlamb, Alan H.; Hunter, William N.
      Molecular Microbiology (2006), 61 (6), 1457-1468CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor detd. at 2.2 Å resoln. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic center are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chem. properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
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    21. 21
      Vickers, T. J.; Beverley, S. M. Folate metabolic pathways in Leishmania. Essays Biochem. 2011, 51, 63– 80,  DOI: 10.1042/bse0510063
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      21
      Folate metabolic pathways in Leishmania
      Vickers, Tim J.; Beverley, Stephen M.
      Essays in Biochemistry (2011), 51 (Molecular Parasitology), 63-80CODEN: ESBIAV; ISSN:0071-1365. (Portland Press Ltd.)
      A review. Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metab. from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for 'repurposing' of compds. developed originally for treatment of human cancers or other infectious agents.
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    22. 22
      Ong, H. B.; Sienkiewicz, N.; Wyllie, S.; Fairlamb, A. H. Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major. J. Biol. Chem. 2011, 286, 10429– 10438,  DOI: 10.1074/jbc.M110.209593
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      22
      Dissecting the Metabolic Roles of Pteridine Reductase 1 in Trypanosoma brucei and Leishmania major
      Ong, Han B.; Sienkiewicz, Natasha; Wyllie, Susan; Fairlamb, Alan H.
      Journal of Biological Chemistry (2011), 286 (12), 10429-10438CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H4B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity assocd. with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiol. pH and ionic strength and compared with LmQDPR. Specificity consts. (kcat/Km) for LmPTR1 are similar with dihydrobiopterin (H2B) and quinonoid dihydrobiopterin (qH2B) as substrates and about 20-fold lower than LmQDPR with qH2B. In contrast, TbPTR1 shows a 10-fold higher kcat/Km for H2B over qH2B. Anal. of Trypanosoma brucei isolated from infected rats revealed that H4B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H4B. Gene knock-out expts. confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H4B as a medium supplement. These cells grew normally with H4B, which spontaneously oxidizes to qH2B, but were unable to survive in the absence of pterin or with either biopterin or H2B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metab. in African trypanosomes and underline its potential as a drug target.
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      Sienkiewicz, N.; Ong, H. B.; Fairlamb, A. H. Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice. Mol. Microbiol. 2010, 77, 658– 671,  DOI: 10.1111/j.1365-2958.2010.07236.x
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      23
      Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice
      Sienkiewicz, Natasha; Ong, Han B.; Fairlamb, Alan H.
      Molecular Microbiology (2010), 77 (3), 658-671CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
      Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metab. in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addn. of tetrahydrobiopterin to cultures. Loss of PTR1 was assocd. with gross morphol. changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased nos. of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localization to the cytosol. Mis-localization was confirmed by digitonin fractionation expts. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.
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    24. 24
      Mpamhanga, C. P.; Spinks, D.; Tulloch, L. B.; Shanks, E. J.; Robinson, D. A.; Collie, I. T.; Fairlamb, A. H.; Wyatt, P. G.; Frearson, J. A.; Hunter, W. N.; Gilbert, I. H.; Brenk, R. One scaffold, three binding modes: Novel and selective pteridine reductase 1 inhibitors derived from fragment hits discovered by virtual screening. J. Med. Chem. 2009, 52, 4454– 4465,  DOI: 10.1021/jm900414x
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      24
      One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening
      Mpamhanga, Chidochangu P.; Spinks, Daniel; Tulloch, Lindsay B.; Shanks, Emma J.; Robinson, David A.; Collie, Iain T.; Fairlamb, Alan H.; Wyatt, Paul G.; Frearson, Julie A.; Hunter, William N.; Gilbert, Ian H.; Brenk, Ruth
      Journal of Medicinal Chemistry (2009), 52 (14), 4454-4465CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The enzyme pteridine reductase 1 (PTR1) is a potential target for new compds. to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chem. series were identified contg. aminobenzothiazole and aminobenzimidazole scaffolds, resp. One of the hits (2-amino-5-chlorobenzimidazole) was subjected to crystal structure anal. and a high resoln. crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogs (2-aminobenzimidazole and 1-(3,4-dichlorobenzyl)-2-aminobenzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein-ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichlorobenzyl)-2-aminobenzimidazole, derivs. were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochem. properties were obtained.
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    25. 25
      Spinks, D.; Ong, H. B.; Mpamhanga, C. P.; Shanks, E. J.; Robinson, D. A.; Collie, I. T.; Read, K. D.; Frearson, J. A.; Wyatt, P. G.; Brenk, R.; Fairlamb, A. H.; Gilbert, I. H. Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1. ChemMedChem. 2011, 6, 302– 308,  DOI: 10.1002/cmdc.201000450
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      25
      Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase 1
      Spinks, Daniel; Ong, Han B.; Mpamhanga, Chidochangu P.; Shanks, Emma J.; Robinson, David A.; Collie, Iain T.; Read, Kevin D.; Frearson, Julie A.; Wyatt, Paul G.; Brenk, Ruth; Fairlamb, Alan H.; Gilbert, Ian H.
      ChemMedChem (2011), 6 (2), 302-308CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimization of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivs. Data are reported on 33 compds. This series was initially discovered by a virtual screening campaign. The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chem. and structure-based approaches, we were able to derive compds. with potent activity against T. brucei PTR1 (Kappi = 7 nM), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compds. displayed weak activity against the parasites. Kinetic studies and anal. indicate that the main reason for the lack of cell potency is due to the compds. having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km = 25 nM and Ki = 2.3 nM, resp.).
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    26. 26
      Cavazzuti, A.; Paglietti, G.; Hunter, W. N.; Gamarro, F.; Piras, S.; Loriga, M.; Allecca, S.; Corona, P.; McLuskey, K.; Tulloch, L.; Gibellini, F.; Ferrari, S.; Costi, M. P. Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1448– 1453,  DOI: 10.1073/pnas.0704384105
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      26
      Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development
      Cavazzuti, Antonio; Paglietti, Giuseppe; Hunter, William N.; Gamarro, Francisco; Piras, Sandra; Loriga, Mario; Alleca, Sergio; Corona, Paola; McLuskey, Karen; Tulloch, Lindsay; Gibellini, Federica; Ferrari, Stefania; Costi, Maria Paola
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (5), 1448-1453CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, a rapid-screening strategy using a folate-based library was combined with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling detd. selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivs., and nine compds. showed greater activity against parasite enzymes compared with human enzymes. Compd. I [R = H, Me (II)] displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:I ternary complex revealed a substrate-like binding mode distinct from that previously obsd. for similar compds. A second round of design, synthesis, and assay produced a compd. II with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also detd. Biol. evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was obsd. when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a redn. in toxicity of treatment was obsd. with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.
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      Ivanetich, K. M.; Santi, D. V. Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa. FASEB J. 1990, 4, 1591– 1597,  DOI: 10.1096/fasebj.4.6.2180768
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      Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa
      Ivanetich, Kathryn M.; Santi, Daniel V.
      FASEB Journal (1990), 4 (6), 1591-7CODEN: FAJOEC; ISSN:0892-6638.
      A review with 50 refs. on the structure and function of the title enzymes in Leishmania, Plasmodium, and other protozoa.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktVejs7Y%253D&md5=5f296c3e837cb139eb1ab957c8aa2fb5
    28. 28
      Schormann, N.; Senkovich, O.; Walker, K.; Wright, D. L.; Anderson, A. C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D. Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function. Proteins 2008, 73, 889– 901,  DOI: 10.1002/prot.22115
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      28
      Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function
      Schormann, N.; Senkovich, O.; Walker, K.; Wright, D. L.; Anderson, A. C.; Rosowsky, A.; Ananthan, S.; Shinkre, B.; Velu, S.; Chattopadhyay, D.
      Proteins: Structure, Function, and Bioinformatics (2008), 73 (4), 889-901CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      We have employed a structure-based three-dimensional quant. structure-activity relationship (3D-QSAR) approach to predict the biochem. activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between exptl. and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compds. (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of addnl. activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.
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    29. 29
      Schormann, N.; Velu, S. E.; Murugesan, S.; Senkovich, O.; Walker, K.; Chenna, B. C.; Shinkre, B.; Desai, A.; Chattopadhyay, D. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase. Bioorg. Med. Chem. 2010, 18, 4056– 4066,  DOI: 10.1016/j.bmc.2010.04.020
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      Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase
      Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish
      Bioorganic & Medicinal Chemistry (2010), 18 (11), 4056-4066CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas' disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compd. was detd. against T. cruzi and human DHFR. One of these compds., Et 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystd. with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was detd. Mol. docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compds. are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the resp. enzymes were calcd. based on the exptl. or docked binding mode. An estd. 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.
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    30. 30
      Corona, P.; Gibellini, F.; Cavalli, A.; Saxena, P.; Carta, A.; Loriga, M.; Luciani, R.; Paglietti, G.; Guerrieri, D.; Nerini, E.; Gupta, S.; Hannaert, V.; Michels, P. A. M.; Ferrari, S.; Costi, P. M. Structure-based selectivity optimization of piperidine-pteridine derivatives as potent Leishmania pteridine reductase inhibitors. J. Med. Chem. 2012, 55, 8318– 8329,  DOI: 10.1021/jm300563f
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      Structure-Based Selectivity Optimization of Piperidine-Pteridine Derivatives as Potent Leishmania Pteridine Reductase Inhibitors
      Corona, Paola; Gibellini, Federica; Cavalli, Andrea; Saxena, Puneet; Carta, Antonio; Loriga, Mario; Luciani, Rosaria; Paglietti, Giuseppe; Guerrieri, Davide; Nerini, Erika; Gupta, Shreedhara; Hannaert, Veronique; Michels, Paul A. M.; Ferrari, Stefania; Costi, Paola M.
      Journal of Medicinal Chemistry (2012), 55 (19), 8318-8329CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compd. library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was obsd. for compd. 5b (I). This work represents a step forward in the identification of effective antileishmania agents.
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    31. 31
      Panecka-Hofman, J.; Pöhner, I.; Spyrakis, F.; Zeppelin, T.; Di Pisa, F.; Dello Iacono, L.; Bonucci, A.; Quotadamo, A.; Venturelli, A.; Mangani, S.; Costi, M. P.; Wade, R. C. Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 3215– 3230,  DOI: 10.1016/j.bbagen.2017.09.012
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      Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors
      Panecka-Hofman, Joanna; Poehner, Ina; Spyrakis, Francesca; Zeppelin, Talia; Di Pisa, Flavio; Dello Iacono, Lucia; Bonucci, Alessio; Quotadamo, Antonio; Venturelli, Alberto; Mangani, Stefano; Costi, Maria Paola; Wade, Rebecca C.
      Biochimica et Biophysica Acta, General Subjects (2017), 1861 (12), 3215-3230CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      Multi-target approaches are necessary to properly analyze or modify the function of a biochem. pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of exptl. validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter. The authors computationally compared the structural, dynamic and physico-chem. properties of the targets. The authors based the anal. on available inhibitory activity and crystallog. data, including a crystal structure of the bifunctional T. cruzi DHFR-TS with tetrahydrofolate bound detd. Due to the low sequence and structural similarity of the targets analyzed, the authors employed a mapping of binding pockets based on the known common ligands, folate and methotrexate. The authors' anal. provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallog. structures. The ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosomatid agents. Apart from the target-based discovery of selective compds., the authors' approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.
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    32. 32
      Tulloch, L. B.; Martini, V. P.; Iulek, J.; Huggan, J. K.; Lee, J. H.; Gibson, C. L.; Smith, T. K.; Suckling, C. J.; Hunter, W. N. Structure-based design of pteridine reductase inhibitors targeting African sleeping sickness and the leishmaniases. J. Med. Chem. 2010, 53, 221– 229,  DOI: 10.1021/jm901059x
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      Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the Leishmaniasis
      Tulloch, Lindsay B.; Martini, Viviane P.; Iulek, Jorge; Huggan, Judith K.; Lee, Jeong Hwan; Gibson, Colin L.; Smith, Terry K.; Suckling, Colin J.; Hunter, William N.
      Journal of Medicinal Chemistry (2010), 53 (1), 221-229CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. The authors adopted a structure-based approach to the design of novel PTR1 inhibitors based on three mol. scaffolds. A series of compds., most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compds. displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a mol. bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. The authors tested two new compds. with known DHFR inhibitors. A synergistic effect was obsd. for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.
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      Quotadamo, A.; Linciano, P.; Costi, M. P.; Venturelli, A. Optimization of N-alkylation in the synthesis of methotrexate and pteridine-based derivatives under microwave-irradiation. ChemistrySelect 2019, 4, 4429– 4433,  DOI: 10.1002/slct.201900721
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      Optimization of N-alkylation in the Synthesis of Methotrexate and Pteridine-based Derivatives Under Microwave-Irradiation
      Quotadamo, Antonio; Linciano, Pasquale; Costi, Maria Paola; Venturelli, Alberto
      ChemistrySelect (2019), 4 (15), 4429-4433CODEN: CHEMUD; ISSN:2365-6549. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A new efficient and improved microwave-assisted lab-scale process for the prepn. of I and congeners II [R = Et, Ph, benzyl, etc.; R1 = H, Et, Ph, benzyl; RR1 = N-morpholinyl, 4-benzylpiperazin-1-yl] were described. Starting from the com. available 2,4-diamino-6-(hydroxymethyl)pteridine (Pt-OH), I was obtained with an overall 94% yield through a three steps procedure. The crucial yield-limiting and time-consuming step of SN2 substitution between halogenated pteridine and nucleophilic arom. amine was taken. The innovative process, conducted under microwave irradn., improved yield and purity and in particular reduced the reaction time from days to 20 min. The optimized protocol was successfully applied to the synthesis of diverse pteridine-based derivs. II and to the prepn. in gram-scale of antiparasitic MTX derivs. I for in-vivo studies. This new optimized synthetic procedure therefore represented a worthy alternative to the current protocols for the prepn. of pteridine-based derivs.
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      Sajiki, H.; Ikawa, T.; Hirota, K. Reductive and catalytic monoalkylation of primary amines using nitriles as an alkylating reagent. Org. Lett. 2004, 6, 4977– 4980,  DOI: 10.1021/ol047871o
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      Reductive and catalytic monoalkylation of primary amines using nitriles as an alkylating reagent
      Sajiki, Hironao; Ikawa, Takashi; Hirota, Kosaku
      Organic Letters (2004), 6 (26), 4977-4980CODEN: ORLEF7; ISSN:1523-7060. (American Chemical Society)
      A selective and catalytic mono-N-alkylation method of both arom. and aliph. amines using nitriles as an alkylating agent with Pd/C or Rh/C as a catalyst is described. This method was particularly attractive to provide an environmentally benign and applicable alkylation method of amines without using toxic and corrosive alkylating agents such as alkyl halides and carbonyl compds.
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      Ikawa, T.; Fujita, Y.; Mizusaki, T.; Betsuin, S.; Takamatsu, H.; Maegawa, T.; Monguchi, Y.; Sajiki, H. Selective N-alkylation of amines using nitriles under hydrogenation conditions: facile synthesis of secondary and tertiary amines. Org. Biomol. Chem. 2012, 10, 293– 304,  DOI: 10.1039/C1OB06303K
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      Selective N-alkylation of amines using nitriles under hydrogenation conditions: facile synthesis of secondary and tertiary amines
      Ikawa, Takashi; Fujita, Yuki; Mizusaki, Tomoteru; Betsuin, Sae; Takamatsu, Haruki; Maegawa, Tomohiro; Monguchi, Yasunari; Sajiki, Hironao
      Organic & Biomolecular Chemistry (2012), 10 (2), 293-304CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Nitriles were found to be highly effective alkylating reagents for the selective N-alkylation of amines under catalytic hydrogenation conditions. For the arom. primary amines, the corresponding secondary amines were selectively obtained under Pd/C-catalyzed hydrogenation conditions. Although the use of electron poor arom. amines or bulky nitriles showed a lower reactivity toward the reductive alkylation, the addn. of NH4OAc enhanced the reactivity to give secondary arom. amines in good to excellent yields. Under the same reaction conditions, arom. nitro compds. instead of the arom. primary amines could be directly transformed into secondary amines via a domino reaction involving the one-pot hydrogenation of the nitro group and the reductive alkylation of the amines. While aliph. amines were effectively converted to the corresponding tertiary amines under Pd/C-catalyzed conditions, Rh/C was a highly effective catalyst for the N-monoalkylation of aliph. primary amines without over-alkylation to the tertiary amines. Furthermore, the combination of the Rh/C-catalyzed N-monoalkylation of the aliph. primary amines and addnl. Pd/C-catalyzed alkylation of the resulting secondary aliph. amines could selectively prep. aliph. tertiary amines possessing three different alkyl groups. According to the mechanistic studies, it seems reasonable to conclude that nitriles were reduced to aldimines before the nucleophilic attack of the amine during the first step of the reaction.
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      Ayedi, M. A.; Le Bigot, Y.; Ammar, H.; Abid, S.; El Gharbi, R.; Delmas, M. Synthesis of primary amines by one-pot reductive amination of aldehydes. Synth. Commun. 2013, 43, 2127– 2133,  DOI: 10.1080/00397911.2012.714830
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      Synthesis of primary amines by one-pot reductive amination of aldehydes
      Ayedi, Mohamed Ali; Le Bigot, Yves; Ammar, Houcine; Abid, Souhir; El Gharbi, Rachid; Delmas, Michel
      Synthetic Communications (2013), 43 (16), 2127-2133CODEN: SYNCAV; ISSN:0039-7911. (Taylor & Francis, Inc.)
      A novel, one-pot, two-step reductive amination of aldehydes for the atom-economical synthesis of primary amines was reported. The amination step was carried out with hydroxylammonium chloride and does not require the use of a base. In the subsequent redn. step, a metal zinc/hydrochloride acid system was used. This method is applicable to both aliph. and arom. aldehydes. The operational simplicity, the short reaction times, and the mild reaction conditions add to the value of this method as a practical alternative to the reductive amination of aldehydes. Go to the publisher's online edition of Synthetic Communications to view the free supplemental file.
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      Gourley, D. G.; Schüttelkopf, A. W.; Leonard, G. A.; Luba, J.; Hardy, L. W.; Beverley, S. M.; Hunter, W. N. Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites. Nat. Struct. Biol. 2001, 8, 521– 525,  DOI: 10.1038/88584
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      Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites
      Gourley, David G.; Schottelkopf, Alexander W.; Leonard, Gordon A.; Luba, James; Hardy, Larry W.; Beverley, Stephen M.; Hunter, William N.
      Nature Structural Biology (2001), 8 (6), 521-525CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)
      Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step redn. of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structure of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first redn. uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.
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      Di Pisa, F.; Landi, G.; Dello Iacono, L.; Pozzi, C.; Borsari, C.; Ferrari, S.; Santucci, M.; Santarem, N.; Cordeiro-da-Silva, A.; Moraes, C. B.; Alcantara, L. M.; Fontana, V.; Freitas-Junior, L. H.; Gul, S.; Kuzikov, M.; Behrens, B.; Pöhner, I.; Wade, R. C.; Costi, M. P.; Mangani, S. Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity. Molecules 2017, 22, 426,  DOI: 10.3390/molecules22030426
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      Chroman-4-one derivatives targeting pteridine reductase 1 and showing anti-parasitic activity
      Di Pisa, Flavio; Landi, Giacomo; Iacono, Lucia Dello; Pozzi, Cecilia; Borsari, Chiara; Ferrari, Stefania; Santucci, Matteo; Santarem, Nuno; Cordeiro-da-Silva, Anabela; Moraes, Carolina B.; Alcantara, Laura M.; Fontana, Vanessa; Freitas, Lucio H., Jr.; Gul, Sheraz; Kuzikov, Maria; Behrens, Birte; Pohner, Ina; Wade, Rebecca C.; Costi, Maria Paola; Mangani, Stefano
      Molecules (2017), 22 (3), 426/1-426/16CODEN: MOLEFW; ISSN:1420-3049. (MDPI AG)
      Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogs (1-3) of previously published chromen-4-one derivs. were synthesized and biol. evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compd. 1 and the first crystal structures of LmPTR1-flavanone complexes (compds. 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivs. was explained by comparison of obsd. and predicted binding modes of the compds. Compd. 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compds. targeting multiple PTR1 variants.
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      Lloyd, M. D. High-Throughput Screening for the Discovery of Enzyme Inhibitors. J. Med. Chem. 2020, 63, 10742– 10772,  DOI: 10.1021/acs.jmedchem.0c00523
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      High-Throughput Screening for the Discovery of Enzyme Inhibitors
      Lloyd, Matthew D.
      Journal of Medicinal Chemistry (2020), 63 (19), 10742-10772CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Enzymes are common targets in high-throughput screening and related campaigns. An anal. of papers published between 1990 and 2018 showed that kinases were the most common enzymes investigated, fluorescence-based assays were the most common readout method, and cancer and bacterial infections were the most common therapeutic areas. High-throughput screening and fragment-screening campaigns published between 2017 and 2019 were analyzed in more depth, giving 75 examples of hit to lead development. Kinases, phosphatases, proteases, and peptidases were the most common targets, fluorescent assays were the most commonly used, and a wide variety of structural features were obsd. within the derived drugs. Hit frequency was largely independent of library size and pos. correlated with Z' value for the assay. Binding of metal ions to library compds. and substrates is an underappreciated source of false-pos. results and unreproducible behavior.
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      Holdgate, G.; Meek, T.; Grimley, R. Mechanistic enzymology in drug discovery: a fresh perspective. Nat. Rev. Drug Discovery 2018, 17, 115– 132,  DOI: 10.1038/nrd.2017.219
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      Mechanistic enzymology in drug discovery: a fresh perspective
      Holdgate, Geoffrey A.; Meek, Thomas D.; Grimley, Rachel L.
      Nature Reviews Drug Discovery (2018), 17 (2), 115-132CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)
      A review. Given the therapeutic and com. success of small-mol. enzyme inhibitors, as exemplified by kinase inhibitors in oncol., a major focus of current drug-discovery and development efforts is on enzyme targets. Understanding the course of an enzyme-catalyzed reaction can help to conceptualize different types of inhibitor and to inform the design of screens to identify desired mechanisms. Exploiting this information allows the thorough evaluation of diverse compds., providing the knowledge required to efficiently optimize leads towards differentiated candidate drugs. This review highlights the rationale for conducting high-quality mechanistic enzymol. studies and considers the added value in combining such studies with orthogonal biophys. methods.
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      Synold, T. W.; Willits, E. M.; Barredo, J. C. Role of folylpolyglutamate synthetase (FPGS) in antifolate chemotherapy; a biochemical and clinical update. Leuk. Lymphoma 1996, 21, 9– 15,  DOI: 10.3109/10428199609067573
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      Role of folylpolygutamate synthetase (FPGS) in antifolate chemotherapy; a biochemical and clinical update
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      Leukemia & lymphoma (1996), 21 (1-2), 9-15 ISSN:1042-8194.
      Even though folate antimetabolites were introduced over forty years ago, they continue to be the backbone of many active chemotherapeutic regimens used by medical and pediatric oncologists. The recognition of polyglutamylation by folylpolyglutamate synthetase (FPGS) as an important metabolic step in the "activation" of classical antifolates and novel drugs aimed at thymidylate synthase (TS) and de novo purine synthesis, has resulted in renewed interest in this class of drugs. In addition, the emergence of secondary neoplasms in patients treated with alkylating agents and topoisomerase inhibitors in contrast to the exceptional safety record of antimetabolites, underscores the need for clinical trials that incorporate new strategies with known active antimetabolites and novel promising agents. In that context, FPGS is an important target for further laboratory and clinical investigations.
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      Schrödinger Release 2015-4: QikProp v4.6, Schrödinger, LLC: New York, NY, 2015.
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      Jorgensen, W. L.; Duffy, E. M. Prediction of drug solubility from Monte Carlo simulations. Bioorg. Med. Chem. Lett. 2000, 10, 1155– 1158,  DOI: 10.1016/S0960-894X(00)00172-4
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      Prediction of drug solubility from Monte Carlo simulations
      Jorgensen, William L.; Duffy, Erin M.
      Bioorganic & Medicinal Chemistry Letters (2000), 10 (11), 1155-1158CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Science Ltd.)
      Monte Carlo statistical mechanics simulations have been carried out for 150 org. solutes in water. Phys. significant descriptors such as the solvent-accessible surface area, nos. of hydrogen bonds, and indexes for cohesive interactions in solids are correlated with pharmacol. important properties including octanol/water partition coeff. (log P) and aq. soly. (log S). The regression equation for log S only requires five descriptors to provide a correlation coeff., r2, of 0.9 and rms error of 0.7 for the 150 solutes. The descriptors can form a basis for structural modifications to guide an analog's properties into desired ranges.
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      Borsari, C.; Luciani, R.; Pozzi, C.; Poehner, I.; Henrich, S.; Trande, M.; Cordeiro-da-Silva, A.; Santarem, N.; Baptista, C.; Tait, A.; Di Pisa, F.; Dello Iacono, L.; Landi, G.; Gul, S.; Wolf, M.; Kuzikov, M.; Ellinger, B.; Reinshagen, J.; Witt, G.; Gribbon, P.; Kohler, M.; Keminer, O.; Behrens, B.; Costantino, L.; Tejera Nevado, P.; Bifeld, E.; Eick, J.; Clos, J.; Torrado, J.; Jiménez-Antón, M. D.; Corral, M. J.; Alunda, J. M.; Pellati, F.; Wade, R. C.; Ferrari, S.; Mangani, S.; Costi, M. P. Profiling of flavonol derivatives for the development of antitrypanosomatidic drugs. J. Med. Chem. 2016, 59, 7598– 7616,  DOI: 10.1021/acs.jmedchem.6b00698
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      Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs
      Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Poehner, Ina; Henrich, Stefan; Trande, Matteo; Cordeiro-da-Silva, Anabela; Santarem, Nuno; Baptista, Catarina; Tait, Annalisa; Di Pisa, Flavio; Dello Iacono, Lucia; Landi, Giacomo; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Reinshagen, Jeanette; Witt, Gesa; Gribbon, Philip; Kohler, Manfred; Keminer, Oliver; Behrens, Birte; Costantino, Luca; TejeraNevado, Paloma; Bifeld, Eugenia; Eick, Julia; Clos, Joachim; Torrado, Juan; Jimenez-Anton, Maria D.; Corral, Maria J.; Alunda, Jose Ma; Pellati, Federica; Wade, Rebecca C.; Ferrari, Stefania; Mangani, Stefano; Costi, Maria Paola
      Journal of Medicinal Chemistry (2016), 59 (16), 7598-7616CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivs. was synthesized. Twelve compds. showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the obsd. structure-activity relationships. Compd. 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compd. 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compd. Combination studies with methotrexate revealed that compd. 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concn. of 1.3 μM, 11.7-fold dose redn. index and no toxicity toward host cells. Our results provide the basis for further chem. modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.
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      Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, O. M.; Ferrari, S.; Marverti, G.; Guerrieri, D.; Ligabue, A.; Frassineti, C.; Pozzi, C.; Mangani, S.; Fessas, D.; Guerrini, R.; Ponterini, G.; Wade, R. C.; Costi, M. P. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, E542– E549,  DOI: 10.1073/pnas.1104829108
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      Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase
      Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; Henrich, Stefan; Salo-Ahen, Outi M. H.; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; Pozzi, Cecilia; Mangani, Stefano; Fessas, Dimitrios; Guerrini, Remo; Ponterini, Glauco; Wade, Rebecca C.; Costi, M. Paola
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (34), E542-E549, SE542/1-SE542/22CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clin. important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concns. in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.
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      Benvenuti, M.; Mangani, S. Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2007, 2, 1633– 1651,  DOI: 10.1038/nprot.2007.198
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      Benvenuti, Manuela; Mangani, Stefano
      Nature Protocols (2007), 2 (7), 1633-1651CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      The prepn. of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biol. macromol. The complete automation of the crystn. procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. Many research projects from departmental labs. are devoted to the study of few specific proteins. Here, the authors try to provide a series of protocols for the crystn. of sol. proteins, esp. the difficult ones, tailored for small-scale research groups. An est. of the time needed to complete each of the steps described can be found at the end of each section.
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      Battye, T. G. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.; Leslie, A. G. W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol Crystallogr 2011, 67, 271– 281,  DOI: 10.1107/S0907444910048675
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      iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM
      Battye, T. Geoff G.; Kontogiannis, Luke; Johnson, Owen; Powell, Harold R.; Leslie, Andrew G. W.
      Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 271-281CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      IMOSFLM is a graphical user interface to the diffraction data-integration program MOSFLM. It is designed to simplify data processing by dividing the process into a series of steps, which are normally carried out sequentially. Each step has its own display pane, allowing control over parameters that influence that step and providing graphical feedback to the user. Suitable values for integration parameters are set automatically, but addnl. menus provide a detailed level of control for experienced users. The image display and the interfaces to the different tasks (indexing, strategy calcn., cell refinement, integration and history) are described. The most important parameters for each step and the best way of assessing success or failure are discussed.
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      Powell, H. R.; Johnson, O.; Leslie, A. G. W. Autoindexing diffraction images with iMosflm. Acta Crystallogr. D Biol Crystallogr 2013, 69, 1195– 1203,  DOI: 10.1107/S0907444912048524
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      Autoindexing diffraction images with iMosflm
      Powell, Harold R.; Johnson, Owen; Leslie, Andrew G. W.
      Acta Crystallographica, Section D: Biological Crystallography (2013), 69 (7), 1195-1203CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. An overview of autoindexing diffraction images based on one-dimensional fast Fourier transforms is presented. The implementation of the algorithm in the Mosflm/iMosflm program suite is described with a discussion of practical issues that may arise and ways of assessing the success or failure of the procedure. Recent developments allow indexing of images that show multiple lattices, and several examples demonstrate the success of this approach in real cases.
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      Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol Crystallogr 2006, 62, 72– 82,  DOI: 10.1107/S0907444905036693
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      Scaling and assessment of data quality
      Evans, Philip
      Acta Crystallographica, Section D: Biological Crystallography (2006), D62 (1), 72-82CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      The various phys. factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analyzed to set the real resoln. of the data set, to detect bad regions (e.g. bad images), to analyze radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation anal. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be detd. from scores such as correlation coeffs. between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.
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      Evans, P. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D Biol Crystallogr. 2011, 67, 282– 292,  DOI: 10.1107/S090744491003982X
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      An introduction to data reduction: Space-group determination, scaling and intensity statistics
      Evans, Philip R.
      Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 282-292CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. This paper presents an overview of how to run the CCP4 programs for data redn. (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers detn. of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examn. of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estn. of |F| from intensity and finally examn. of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups.
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      CCP4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol Crystallogr. 1994, 50, 760– 763,  DOI: 10.1107/S0907444994003112
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      Vagin, A.; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol Crystallogr. 2010, 66, 22– 25,  DOI: 10.1107/S0907444909042589
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      Molecular replacement with MOLREP
      Vagin, Alexei; Teplyakov, Alexei
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 22-25CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.
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      Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol Crystallogr 2011, 67, 355– 367,  DOI: 10.1107/S0907444911001314
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      REFMAC5 for the refinement of macromolecular crystal structures
      Murshudov, Garib N.; Skubak, Pavol; Lebedev, Andrey A.; Pannu, Navraj S.; Steiner, Roberto A.; Nicholls, Robert A.; Winn, Martyn D.; Long, Fei; Vagin, Alexei A.
      Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 355-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      This paper describes various components of the macromol. crystallog. refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS exptl. diffraction data. To ensure chem. and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolns. at least as low as 4 Å can be achieved thanks to low-resoln. refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, 'jelly-body' restraints and the use of novel long-range restraints on at. displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 addnl. offers TLS parameterization and, when high-resoln. data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resoln. spectrum encountered in macromol. crystallog.
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      Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol Crystallogr 2004, 60, 2126– 2132,  DOI: 10.1107/S0907444904019158
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      Coot: model-building tools for molecular graphics
      Emsley, Paul; Cowtan, Kevin
      Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
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      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol Crystallogr 2010, 66, 486– 501,  DOI: 10.1107/S0907444910007493
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      Features and development of Coot
      Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
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      Langer, G. G.; Cohen, S. X.; Lamzin, V. S.; Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 2008, 3, 1171– 1179,  DOI: 10.1038/nprot.2008.91
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      Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7
      Langer, Gerrit; Cohen, Serge X.; Lamzin, Victor S.; Perrakis, Anastassis
      Nature Protocols (2008), 3 (7), 1171-1179CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      ARP/wARP is a software suite to build macromol. models in x-ray crystallog. electron d. maps. Structural genomics initiatives and the study of complex macromol. assemblies and membrane proteins all rely on advanced methods for 3D structure detn. ARP/wARP meets these needs by providing the tools to obtain a macromol. model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water mols. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.
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      Laskowski, R. A.; MacArthur, M. W.; Thornton, J. M. Validation of protein models derived from experiment. Curr. Opin. Struct. Biol. 1998, 8, 631– 639,  DOI: 10.1016/S0959-440X(98)80156-5
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      Validation of protein models derived from experiment
      Laskowski, Roman A.; MacArthur, Malcolm W.; Thornton, Janet M.
      Current Opinion in Structural Biology (1998), 8 (5), 631-639CODEN: COSBEF; ISSN:0959-440X. (Current Biology Publications)
      A review with 55 refs. The growing no. of protein structures solved at at. resoln. holds the promise of further improvements in geometry-based validation parameters. Addnl., the estd. std. uncertainties of the at. coordinates have been computed for a no. of x-ray structures, providing a measure of the coordinate precision. In NMR spectroscopy, a measure analogous to the crystallog. R-factor has been developed.
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      Potterton, L.; McNicholas, S.; Krissinel, E.; Gruber, J.; Cowtan, K.; Emsley, P.; Murshudov, G. N.; Cohen, S.; Perrakis, A.; Noble, M. Developments in the CCP4 molecular-graphics project. Acta Crystallogr. D Biol Crystallogr 2004, 60, 2288– 2294,  DOI: 10.1107/S0907444904023716
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      Developments in the CCP4 molecular-graphics project
      Potterton, Liz; McNicholas, Stuart; Krissinel, Eugene; Gruber, Jan; Cowtan, Kevin; Emsley, Paul; Murshudov, Garib N.; Cohen, Serge; Perrakis, Anastassis; Noble, Martin
      Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2288-2294CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      Progress towards structure detn. that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-d. map interpretation and for the anal. of the resulting at. models. Advances in map-interpretation algorithms are extending the resoln. regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromol. electron d., particularly where crystallog. data is only available to modest resoln. [for example, I/σ(I) < 2.0 for min. resoln. 2.5 Å]. In such cases, a set of manual and semi-manual model-building mol.-graphics tools is needed. At the same time, converting the knowledge encapsulated in a mol. structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by both static and dynamic representations. CCP4mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biol. As well as providing a carefully designed user interface to advanced algorithms of model building and anal., CCP4mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVars7vN&md5=6bdc27bb8dc2d7c5ed37ef306e686001
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      Shanks, E. J.; Ong, H. B.; Robinson, D. A.; Thompson, S.; Sienkiewicz, N.; Fairlamb, A. H.; Frearson, J. A. Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase. Anal. Biochem. 2010, 396, 194– 203,  DOI: 10.1016/j.ab.2009.09.003
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      Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase
      Shanks, Emma J.; Ong, Han B.; Robinson, David A.; Thompson, Stephen; Sienkiewicz, Natasha; Fairlamb, Alan H.; Frearson, Julie A.
      Analytical Biochemistry (2010), 396 (2), 194-203CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidn. of NADP (NADPH). Although this assay has been adequate to study the biol. of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymically coupled screening assay in which the product of the enzymic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-mol. inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFShtb3N&md5=73a55624db0bf7bc04b768bef03807df
    61. 61
      Ferrari, S.; Morandi, F.; Motiejunas, D.; Nerini, E.; Henrich, S.; Luciani, R.; Venturelli, A.; Lazzari, S.; Calò, S.; Gupta, S.; Hannaert, V.; Michels, P. A. M.; Wade, R. C.; Costi, M. P. Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase. J. Med. Chem. 2011, 54, 211– 221,  DOI: 10.1021/jm1010572
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      Virtual Screening Identification of Nonfolate Compounds, Including a CNS Drug, as Antiparasitic Agents Inhibiting Pteridine Reductase
      Ferrari, Stefania; Morandi, Federica; Motiejunas, Domantas; Nerini, Erika; Henrich, Stefan; Luciani, Rosaria; Venturelli, Alberto; Lazzari, Sandra; Calo, Samuele; Gupta, Shreedhara; Hannaert, Veronique; Michels, Paul A. M.; Wade, Rebecca C.; Costi, M. Paola
      Journal of Medicinal Chemistry (2011), 54 (1), 211-221CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Folate analog inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new mols. with specificity for PTR1, we carried out a virtual screening of the Available Chems. Directory (ACD) database to select compds. that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like mols. with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compds. both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compds. showed efficacy only in combination. In toxicity tests against human fibroblasts, several compds. showed low toxicity. One compd., 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclin. evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.
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      Tan, X.; Huang, S.; Ratnam, M.; Thompson, P. D.; Freisheim, J. H. The importance of loop region residues 40–46 in human dihydrofolate reductase as revealed by site-directed mutagenesis. J. Biol. Chem. 1990, 265, 8027– 8032,  DOI: 10.1016/S0021-9258(19)39034-9
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      The importance of loop region residues 40-46 in human dihydrofolate reductase as revealed by site-directed mutagenesis
      Tan, Xuehai; Huang, Shaoming; Ratnam, Manohar; Thompson, Paul D.; Freisheim, James H.
      Journal of Biological Chemistry (1990), 265 (14), 8027-32CODEN: JBCHA3; ISSN:0021-9258.
      Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible loop region between residues 40 and 46 of human dihydrofolate reductase. Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and NADPH as well as the pH rate profile are virtually identical to that of the wild type. In contrast, the Vmax of the mutant enzyme is decreased 2.5-fold. The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates. Agents such as KCl, urea, and organomercurials at concns. which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant. Competitive ELISA expts. using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking redn. in the binding of the antibodies. Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies. The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus communicating the effects of ligand binding.
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      Schrödinger Release 2015-4: LigPrep v3.6; Epik v3.4; Protein Preparation Wizard (PrepWizard); SiteMap v3.7; Glide v6.9; Induced Fit Docking Protocol; Prime v4.2, Schrödinger, LLC: New York, NY, 2015.
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      Linciano, P.; Dawson, A.; Pöhner, I.; Costa, D. M.; Sá, M. S.; Cordeiro-da-Silva, A.; Luciani, R.; Gul, S.; Witt, G.; Ellinger, B.; Kuzikov, M.; Gribbon, P.; Reinshagen, J.; Wolf, M.; Behrens, B.; Hannaert, V.; Michels, P. A. M.; Nerini, E.; Pozzi, C.; Di Pisa, F.; Landi, G.; Santarem, N.; Ferrari, S.; Saxena, P.; Lazzari, S.; Cannazza, G.; Freitas-Junior, L. H.; Moraes, C. B.; Pascoalino, B. S.; Alcântara, L. M.; Bertolacini, C. P.; Fontana, V.; Wittig, U.; Müller, W.; Wade, R. C.; Hunter, W. N.; Mangani, S.; Costantino, L.; Costi, M. P. Exploiting the 2-Amino-1,3,4-thiadiazole scaffold to inhibit Trypanosoma brucei pteridine reductase in support of early-stage drug discovery. ACS Omega 2017, 2, 5666– 5683,  DOI: 10.1021/acsomega.7b00473
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      Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery
      Linciano, Pasquale; Dawson, Alice; Pohner, Ina; Costa, David M.; Sa, Monica S.; Cordeiro-da-Silva, Anabela; Luciani, Rosaria; Gul, Sheraz; Witt, Gesa; Ellinger, Bernhard; Kuzikov, Maria; Gribbon, Philip; Reinshagen, Jeanette; Wolf, Markus; Behrens, Birte; Hannaert, Veronique; Michels, Paul A. M.; Nerini, Erika; Pozzi, Cecilia; di Pisa, Flavio; Landi, Giacomo; Santarem, Nuno; Ferrari, Stefania; Saxena, Puneet; Lazzari, Sandra; Cannazza, Giuseppe; Freitas-Junior, Lucio H.; Moraes, Carolina B.; Pascoalino, Bruno S.; Alcantara, Laura M.; Bertolacini, Claudia P.; Fontana, Vanessa; Wittig, Ulrike; Muller, Wolfgang; Wade, Rebecca C.; Hunter, William N.; Mangani, Stefano; Costantino, Luca; Costi, Maria P.
      ACS Omega (2017), 2 (9), 5666-5683CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. The authors investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). The authors detd. crystal structures of several TbPTR1-inhibitor complexes to guide structure-based design of new thiadiazole derivs. Subsequent synthesis, enzyme and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compd. I, a biphenyl-thiadiazole-2,5-diamine, with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase (DHFR) inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. The antiparasitic activity of the (I + MTX) combination was reversed by addn. of folic acid. By adopting an efficient early hit discovery platform, the authors demonstrate with the 2-amino-1,3,4-thiadiazole scaffold how a promising tool for the development of anti-Trypanosoma brucei agents can be obtained.
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      Shelley, J. C.; Cholleti, A.; Frye, L. L.; Greenwood, J. R.; Timlin, M. R.; Uchimaya, M. Epik: a software program for pKa prediction and protonation state generation for drug-like molecules. J. Comput. Aided Mol. Des. 2007, 21, 681– 691,  DOI: 10.1007/s10822-007-9133-z
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      Epik: a software program for pKa prediction and protonation state generation for drug-like molecules
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      Journal of Computer-Aided Molecular Design (2007), 21 (12), 681-691CODEN: JCADEQ; ISSN:0920-654X. (Springer)
      Epik is a computer program for predicting pKa values for drug-like mols. Epik can use this capability in combination with technol. for tautomerization to adjust the protonation state of small drug-like mols. to automatically generate one or more of the most probable forms for use in further mol. modeling studies. Many medicinal chems. can exchange protons with their environment, resulting in various ionization and tautomeric states, collectively known as protonation states. The protonation state of a drug can affect its soly. and membrane permeability. In modeling, the protonation state of a ligand will also affect which conformations are predicted for the mol., as well as predictions for binding modes and ligand affinities based upon protein-ligand interactions. Despite the importance of the protonation state, many databases of candidate mols. used in drug development do not store reliable information on the most probable protonation states. Epik is sufficiently rapid and accurate to process large databases of drug-like mols. to provide this information. Several new technologies are employed. Extensions to the well-established Hammett and Taft approaches are used for pKa prediction, namely, mesomer standardization, charge cancellation, and charge spreading to make the predicted results reflect the nature of the mol. itself rather just for the particular Lewis structure used on input. In addn., a new iterative technol. for generating, ranking and culling the generated protonation states is employed.
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      Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J. Comput. Aided Mol. Des. 2010, 24, 591– 604,  DOI: 10.1007/s10822-010-9349-1
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      Greenwood, Jeremy R.; Calkins, David; Sullivan, Arron P.; Shelley, John C.
      Journal of Computer-Aided Molecular Design (2010), 24 (6-7), 591-604CODEN: JCADEQ; ISSN:0920-654X. (Springer)
      A review. Generating the appropriate protonation states of drug-like mols. in soln. is important for success in both ligand- and structure-based virtual screening. Screening collections of millions of compds. requires a method for detg. tautomers and their energies that is sufficiently rapid, accurate, and comprehensive. To maximize enrichment, the lowest energy tautomers must be detd. from heterogeneous input, without over-enumerating unfavorable states. While computationally expensive, the d. functional theory (DFT) method M06-2X/aug-cc-pVTZ(-f) [PB-SCRF] provides accurate energies for enumerated model tautomeric systems. The empirical Hammett-Taft methodol. can very rapidly extrapolate substituent effects from model systems to drug-like mols. via the relationship between pKT and pKa. Combining the 2 complementary approaches transforms the tautomer problem from a scientific challenge to one of engineering scale-up, and avoids issues that arise due to the very limited no. of measured pKT values, esp. for the complicated heterocycles often favored by medicinal chemists for their novelty and versatility. Several hundreds of pre-calcd. tautomer energies and substituent pKa effects are tabulated in databases for use in structural adjustment by the program Epik, which treats tautomers as a subset of the larger problem of the protonation states in aq. ensembles and their energy penalties. Accuracy and coverage is continually improved and expanded by parameterizing new systems of interest using DFT and exptl. data. Recommendations are made for how to best incorporate tautomers in mol. design and virtual screening workflows.
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      Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity
      Sanschagrin, Paul C.; Kuhn, Leslie A.
      Protein Science (1998), 7 (10), 2054-2064CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)
      Cluster anal. is presented as a technique for analyzing the conservation and chem. of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster anal. provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water mols., and also avoids reliance on any one structure as a ref. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water mols. from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concd. in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is assocd. with ligand selectivity. Cluster anal. also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addn. to facilitating the anal. of water sites from multiple structures, cluster anal. provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.
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      Madhavi Sastry, G.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 2013, 27, 221– 234,  DOI: 10.1007/s10822-013-9644-8
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      Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments
      Madhavi Sastry, G.; Adzhigirey, Matvey; Day, Tyler; Annabhimoju, Ramakrishna; Sherman, Woody
      Journal of Computer-Aided Molecular Design (2013), 27 (3), 221-234CODEN: JCADEQ; ISSN:0920-654X. (Springer)
      Structure-based virtual screening plays an important role in drug discovery and complements other screening approaches. In general, protein crystal structures are prepd. prior to docking in order to add hydrogen atoms, optimize hydrogen bonds, remove at. clashes, and perform other operations that are not part of the x-ray crystal structure refinement process. In addn., ligands must be prepd. to create 3-dimensional geometries, assign proper bond orders, and generate accessible tautomer and ionization states prior to virtual screening. While the prerequisite for proper system prepn. is generally accepted in the field, an extensive study of the prepn. steps and their effect on virtual screening enrichments has not been performed. In this work, we systematically explore each of the steps involved in prepg. a system for virtual screening. We first explore a large no. of parameters using the Glide validation set of 36 crystal structures and 1,000 decoys. We then apply a subset of protocols to the DUD database. We show that database enrichment is improved with proper prepn. and that neglecting certain steps of the prepn. process produces a systematic degrdn. in enrichments, which can be large for some targets. We provide examples illustrating the structural changes introduced by the prepn. that impact database enrichment. While the work presented here was performed with the Protein Prepn. Wizard and Glide, the insights and guidance are expected to be generalizable to structure-based virtual screening with other docking methods.
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      Li, H.; Robertson, A. D.; Jensen, J. H. Very fast empirical prediction and rationalization of protein pKa values. Proteins 2005, 61, 704– 721,  DOI: 10.1002/prot.20660
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      Proteins: Structure, Function, and Bioinformatics (2005), 61 (4), 704-721CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      A very fast empirical method is presented for structure-based protein pKa prediction and rationalization. The desolvation effects and intra-protein interactions, which cause variations in pKa values of protein ionizable groups, are empirically related to the positions and chem. nature of the groups proximate to the pKa sites. A computer program is written to automatically predict pKa values based on these empirical relationships within a couple of seconds. Unusual pKa values at buried active sites, which are among the most interesting protein pKa values, are predicted very well with the empirical method. A test on 233 carboxyl, 12 cysteine, 45 histidine, and 24 lysine pKa values in various proteins shows a root-mean-square deviation (RMSD) of 0.89 from exptl. values. Removal of the 29 pKa values that are upper or lower limits results in an RMSD = 0.79 for the remaining 285 pKa values.
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      Banks, J. L.; Beard, H. S.; Cao, Y.; Cho, A. E.; Damm, W.; Farid, R.; Felts, A. K.; Halgren, T. A.; Mainz, D. T.; Maple, J. R.; Murphy, R.; Philipp, D. M.; Repasky, M. P.; Zhang, L. Y.; Berne, B. J.; Friesner, R. A.; Gallicchio, E.; Levy, R. M. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J. Comput. Chem. 2005, 26, 1752– 1780,  DOI: 10.1002/jcc.20292
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      A review. We provide an overview of the IMPACT mol. mechanics program with an emphasis on recent developments and a description of its current functionality. With respect to core mol. mechanics technologies we include a status report for the fixed charge and polarizable force fields that can be used with the program and illustrate how the force fields, when used together with new atom typing and parameter assignment modules, have greatly expanded the coverage of org. compds. and medicinally relevant ligands. As we discuss in this review, explicit solvent simulations have been used to guide our design of implicit solvent models based on the generalized Born framework and a novel nonpolar estimator that have recently been incorporated into the program. With IMPACT it is possible to use several different advanced conformational sampling algorithms based on combining features of mol. dynamics and Monte Carlo simulations. The program includes two specialized mol. mechanics modules: Glide, a high-throughput docking program, and QSite, a mixed quantum mechanics/mol. mechanics module. These modules employ the IMPACT infrastructure as a starting point for the construction of the protein model and assignment of mol. mechanics parameters, but have then been developed to meet specialized objectives with respect to sampling and the energy function.
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      Halgren, T. New method for fast and accurate binding-site identification and analysis. Chem. Biol. Drug Des. 2007, 69, 146– 148,  DOI: 10.1111/j.1747-0285.2007.00483.x
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      New method for fast and accurate binding-site identification and analysis
      Halgren, Tom
      Chemical Biology & Drug Design (2007), 69 (2), 146-148CODEN: CBDDAL; ISSN:1747-0277. (Blackwell Publishing Ltd.)
      Structure-based drug design seeks to exploit the structure of protein-ligand or protein-protein binding sites, but the site is not always known at the outset. Even when the site is known, the researcher may wish to identify alternative prospective binding sites that may result in different biol. effects or new class of compds. It is also vital in lead optimization to clearly understand the degree to which known binders or docking hits satisfy or violate complementarity to the receptor. SiteMap is a new technique for identifying potential binding sites and for predicting their druggability in lead-discovery applications and for characterizing binding sites and critically assessing prospective ligands in lead-optimization applications. In large-scale validation tests, SiteMap correctly identifies the known binding site in > 96% of the cases, with best results (> 98%) coming for sites that bind ligands tightly. It also accurately distinguishes between sites that bind ligands and sites that don't. In binding-site anal., SiteMap provides a wealth of quant. and graphical information that can help guide efforts to modify ligand structure to enhance potency or improve phys. properties. These attributes allow SiteMap to nicely complement techniques such as docking and computational lead optimization in structure-base drug design.
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      Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 2009, 49, 377– 389,  DOI: 10.1021/ci800324m
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      Identifying and Characterizing Binding Sites and Assessing Druggability
      Halgren, Thomas A.
      Journal of Chemical Information and Modeling (2009), 49 (2), 377-389CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      Identification and characterization of binding sites is key in the process of structure-based drug design. In some cases there may not be any information about the binding site for a target of interest. In other cases, a putative binding site has been identified by computational or exptl. means, but the druggability of the target is not known. Even when a site for a given target is known, it may be desirable to find addnl. sites whose targeting could produce a desired biol. response. A new program, called SiteMap, is presented for identifying and analyzing binding sites and for predicting target druggability. In a large-scale validation, SiteMap correctly identifies the known binding site as the top-ranked site in 86% of the cases, with best results (>98%) coming for sites that bind ligands with subnanomolar affinity. In addn., a modified version of the score employed for binding-site identification allows SiteMap to accurately classify the druggability of proteins as measured by their ability to bind passively absorbed small mols. tightly. In characterizing binding sites, SiteMap provides quant. and graphical information that can help guide efforts to critically assess virtual hits in a lead-discovery application or to modify ligand structure to enhance potency or improve phys. properties in a lead-optimization context.
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      Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739– 1749,  DOI: 10.1021/jm0306430
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      Glide: A new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy
      Friesner, Richard A.; Banks, Jay L.; Murphy, Robert B.; Halgren, Thomas A.; Klicic, Jasna J.; Mainz, Daniel T.; Repasky, Matthew P.; Knoll, Eric H.; Shelley, Mee; Perry, Jason K.; Shaw, David E.; Francis, Perry; Shenkin, Peter S.
      Journal of Medicinal Chemistry (2004), 47 (7), 1739-1749CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Unlike other methods for docking ligands to the rigid 3D structure of a known protein receptor, Glide approximates a complete systematic search of the conformational, orientational, and positional space of the docked ligand. In this search, an initial rough positioning and scoring phase that dramatically narrows the search space is followed by torsionally flexible energy optimization on an OPLS-AA nonbonded potential grid for a few hundred surviving candidate poses. The very best candidates are further refined via a Monte Carlo sampling of pose conformation; in some cases, this is crucial to obtaining an accurate docked pose. Selection of the best docked pose uses a model energy function that combines empirical and force-field-based terms. Docking accuracy is assessed by redocking ligands from 282 cocrystd. PDB complexes starting from conformationally optimized ligand geometries that bear no memory of the correctly docked pose. Errors in geometry for the top-ranked pose are less than 1 Å in nearly half of the cases and are greater than 2 Å in only about one-third of them. Comparisons to published data on rms deviations show that Glide is nearly twice as accurate as GOLD and more than twice as accurate as FlexX for ligands having up to 20 rotatable bonds. Glide is also found to be more accurate than the recently described Surflex method.
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      Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 2004, 47, 1750– 1759,  DOI: 10.1021/jm030644s
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      Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening
      Halgren, Thomas A.; Murphy, Robert B.; Friesner, Richard A.; Beard, Hege S.; Frye, Leah L.; Pollard, W. Thomas; Banks, Jay L.
      Journal of Medicinal Chemistry (2004), 47 (7), 1750-1759CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Glide's ability to identify active compds. in a database screen is characterized by applying Glide to a diverse set of nine protein receptors. In many cases, two, or even three, protein sites are employed to probe the sensitivity of the results to the site geometry. To make the database screens as realistic as possible, the screens use sets of "druglike" decoy ligands that have been selected to be representative of what we believe is likely to be found in the compd. collection of a pharmaceutical or biotechnol. company. Results are presented for releases 1.8, 2.0, and 2.5 of Glide. The comparisons show that av. measures for both "early" and "global" enrichment for Glide 2.5 are 3 times higher than for Glide 1.8 and more than 2 times higher than for Glide 2.0 because of better results for the least well-handled screens. This improvement in enrichment stems largely from the better balance of the more widely parametrized GlideScore 2.5 function and the inclusion of terms that penalize ligand-protein interactions that violate established principles of phys. chem., particularly as it concerns the exposure to solvent of charged protein and ligand groups. Comparisons to results for the thymidine kinase and estrogen receptors published by Rognan and co-workers (J. Med. Chem. 2000, 43, 4759-4767) show that Glide 2.5 performs better than GOLD 1.1, FlexX 1.8, or DOCK 4.01.
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    75. 75
      Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. Extra Precision Glide: Docking and scoring incorporating a model of hydrophobic wnclosure for protein-ligand complexes. J. Med. Chem. 2006, 49, 6177– 6196,  DOI: 10.1021/jm051256o
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      Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes
      Friesner, Richard A.; Murphy, Robert B.; Repasky, Matthew P.; Frye, Leah L.; Greenwood, Jeremy R.; Halgren, Thomas A.; Sanschagrin, Paul C.; Mainz, Daniel T.
      Journal of Medicinal Chemistry (2006), 49 (21), 6177-6196CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A novel scoring function to est. protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addn. to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included:(1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce exptl. binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, resp.) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP mol. recognition and water scoring in sepg. active and inactive ligands and avoiding false positives.
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    76. 76
      Sherman, W.; Beard, H. S.; Farid, R. Use of an induced fit receptor structure in virtual screening. Chem. Biol. Drug Des. 2006, 67, 83– 84,  DOI: 10.1111/j.1747-0285.2005.00327.x
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      Use of an induced fit receptor structure in virtual screening
      Sherman, Woody; Beard, Hege S.; Farid, Ramy
      Chemical Biology & Drug Design (2006), 67 (1), 83-84CODEN: CBDDAL; ISSN:1747-0277. (Blackwell Publishing Ltd.)
      A review. The automated induced fit docking protocol was used to generate the DFG-out conformation from a p38 MAP kinase activation loop starting from a DFG-in structure (1a9u) and the ligand from 1kv1 (BMU). In a virtual screening study of 25K decoy ligands and 46 known actives, using an ensemble consisting of the induced fit docking structure (DFG-out) and the 1a9u crystal structure (DFG-in), 14 actives were identified in the top 1% of the database, including BMU and BIRB 796. 3 Actives were identified when 1a9u was used alone.
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    77. 77
      Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. Novel procedure for modeling ligand/receptor induced fit effects. J. Med. Chem. 2006, 49, 534– 553,  DOI: 10.1021/jm050540c
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      77
      Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects
      Sherman, Woody; Day, Tyler; Jacobson, Matthew P.; Friesner, Richard A.; Farid, Ramy
      Journal of Medicinal Chemistry (2006), 49 (2), 534-553CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We present a novel protein-ligand docking method that accurately accounts for both ligand and receptor flexibility by iteratively combining rigid receptor docking (Glide) with protein structure prediction (Prime) techniques. While traditional rigid-receptor docking methods are useful when the receptor structure does not change substantially upon ligand binding, success is limited when the protein must be "induced" into the correct binding conformation for a given ligand. We provide an in-depth description of our novel methodol. and present results for 21 pharmaceutically relevant examples. Traditional rigid-receptor docking for these 21 cases yields an av. RMSD of 5.5 Å. The av. ligand RMSD for docking to a flexible receptor for the 21 pairs is 1.4 Å; the RMSD is ≤1.8 Å for 18 of the cases. For the three cases with RMSDs greater than 1.8 Å, the core of the ligand is properly docked and all key protein/ligand interactions are captured.
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    78. 78
      Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J. F.; Honig, B.; Shaw, D. E.; Friesner, R. A. A hierarchical approach to all-atom protein loop prediction. Proteins 2004, 55, 351– 367,  DOI: 10.1002/prot.10613
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      A hierarchical approach to all-atom protein loop prediction
      Jacobson, Matthew P.; Pincus, David L.; Rapp, Chaya S.; Day, Tyler J. F.; Honig, Barry; Shaw, David E.; Friesner, Richard A.
      Proteins: Structure, Function, and Bioinformatics (2004), 55 (2), 351-367CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      The application of all-atom force fields (and explicit or implicit solvent models) to protein homol.-modeling tasks such as side-chain and loop prediction remains challenging both because of the expense of the individual energy calcns. and because of the difficulty of sampling the rugged all-atom energy surface. Here the authors address this challenge for the problem of loop prediction through the development of numerous new algorithms, with an emphasis on multiscale and hierarchical techniques. As a first step in evaluating the performance of the authors' loop prediction algorithm, the authors have applied it to the problem of reconstructing loops in native structures; the authors also explicitly include crystal packing to provide a fair comparison with crystal structures. In brief, large nos. of loops are generated by using a dihedral angle-based buildup procedure followed by iterative cycles of clustering, side-chain optimization, and complete energy minimization of selected loop structures. The authors evaluate this method by the largest test set yet used for validation of a loop prediction method, with a total of 833 loops ranging from 4 to 12 residues in length. Av./median backbone root-mean-square deviations (RMSDs) to the native structures (superimposing the body of the protein, not the loop itself) are 0.42/0.24 Å for 5 residue loops, 1.00/0.44 Å for 8 residue loops, and 2.47/1.83 Å for 11 residue loops. Median RMSDs are substantially lower than the avs. because of a small no. of outliers; the causes of these failures are examd. in some detail, and many can be attributed to errors in assignment of protonation states of titratable residues, omission of ligands from the simulation, and, in a few cases, probable errors in the exptl. detd. structures. When these obvious problems in the data sets are filtered out, av. RMSDs to the native structures improve to 0.43 Å for 5 residue loops, 0.84 Å for 8 residue loops, and 1.63 Å for 11 residue loops. In the vast majority of cases, the method locates energy min. that are lower than or equal to that of the minimized native loop, thus indicating that sampling rarely limits prediction accuracy. The overall results are, to the authors' knowledge, the best reported to date, and the authors attribute this success to the combination of an accurate all-atom energy function, efficient methods for loop buildup and side-chain optimization, and, esp. for the longer loops, the hierarchical refinement protocol.
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      Jacobson, M. P.; Friesner, R. A.; Xiang, Z.; Honig, B. On the role of the crystal environment in determining protein side-chain conformations. J. Mol. Biol. 2002, 320, 597– 608,  DOI: 10.1016/S0022-2836(02)00470-9
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      79
      On the Role of the Crystal Environment in Determining Protein Side-chain Conformations
      Jacobson, Matthew P.; Friesner, Richard A.; Xiang, Zhexin; Honig, Barry
      Journal of Molecular Biology (2002), 320 (3), 597-608CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Science Ltd.)
      The role of crystal packing in detg. the obsd. conformations of amino acid side-chains in protein crystals is investigated by (1) anal. of a database of proteins that have been crystd. in different unit cells (space group or unit cell dimensions) and (2) theor. predictions of side-chain conformations with the crystal environment explicitly represented. Both of these approaches indicate that the crystal environment plays an important role in detg. the conformations of polar side-chains on the surfaces of proteins. Inclusion of the crystal environment permits a more sensitive measurement of the achievable accuracy of side-chain prediction programs, when validating against structures obtained by x-ray crystallog. Our side-chain prediction program uses an all-atom force field and a Generalized Born model of solvation and is thus capable of modeling simple packing effects (i.e. van der Waals interactions), electrostatic effects, and desolvation, which are all important mechanisms by which the crystal environment impacts obsd. side-chain conformations. Our results are also relevant to the understanding of changes in side-chain conformation that may result from ligand docking and protein-protein assocn., insofar as the results reveal how side-chain conformations change in response to their local environment.
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    80. 80
      Lagorce, D.; Sperandio, O.; Baell, J. B.; Miteva, M. A.; Villoutreix, B. O. FAF-Drugs3: a web server for compound property calculation and chemical library design. Nucleic Acids Res. 2015, 43, W200– W207,  DOI: 10.1093/nar/gkv353
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      FAF-Drugs3: a web server for compound property calculation and chemical library design
      Lagorce, David; Sperandio, Olivier; Baell, Jonathan B.; Miteva, Maria A.; Villoutreix, Bruno O.
      Nucleic Acids Research (2015), 43 (W1), W200-W207CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A review. Drug attrition late in preclin. or clin. development is a serious economic problem in the field of drug discovery. These problems can be linked, in part, to the quality of the compd. collections used during the hit generation stage and to the selection of compds. undergoing optimization. Here, we present FAF-Drugs3, a web server that can be used for drug discovery and chem. biol. projects to help in prepg. compd. libraries and to assist decision-makin