Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Med. Chem. 2016, 59, 16, 7598-7616

Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

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† Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy

‡ Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy

§ Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany

∥ Instituto de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal

⊥ Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany

# Complutense University of Madrid, 28040 Madrid, Spain

∇ Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, 69120 Heidelberg, Germany

○ Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University,69120 Heidelberg, Germany

◆ Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany

¶1 Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain

*For M.P.C.: phone, 0039-059-205-8579; E-mail, [email protected]

*For S.M.: phone, 0039-057-723-4255, 0039-057-723-4252; E-mail, [email protected]

*For S.F.: phone, 0039-059-205-8578; E-mail, [email protected]

Cite this: J. Med. Chem. 2016, 59, 16, 7598–7616

Publication Date (Web):July 14, 2016

https://doi.org/10.1021/acs.jmedchem.6b00698

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Abstract

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 derivatives was synthesized. Twelve compounds showed EC₅₀ values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure–activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Introduction

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Protozoan parasites of the Trypanosomatidae family are the etiological agents of several significant neglected tropical diseases including human African trypanosomiasis (HAT), Chagas’ disease, and leishmaniasis, which collectively affect nearly 10 million people worldwide. HAT is caused by the bloodstream form of Trypanosoma brucei. (1)Leishmania spp. infect macrophages and cause a wide spectrum of symptoms ranging from cutaneous lesions to potentially fatal visceral infections. (2)Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an obligate intracellular parasite that invades different internal organs. Since effective vaccines are lacking, the control of these diseases is based on vector control and chemotherapy. However, the drugs currently available are characterized by high toxicity, limited efficacy, and require a long period of treatment. The first line drugs have been used for over half a century, and their efficacy is compromised by widespread resistance. (3-5) Therefore, there is an urgent requirement for new, safe, and effective drugs. The drug discovery process against these parasites is hampered by the intrinsic biology of these organisms that can include intracellular stages or central nervous system involvement. (6)

Although there are very few validated drug targets for leishmaniasis and HAT, a target-based approach is extensively used in the drug discovery process for neglected tropical diseases. (7) The folate pathway is an established target for the treatment of bacterial infections and some parasitic diseases such as malaria. (8) Drugs targeting folate-dependent enzymes represent useful candidates against trypanosomatidic infections and key enzymes of the folate metabolism are thymidylate synthase (TS) and dihydrofolate reductase (DHFR), which is the target of the antifolate drugs methotrexate (MTX), pyrimethamine (PYR), and trimethoprim (TMP). However, the activity of classical inhibitors of DHFR against Leishmania and Trypanosoma is reduced because of pteridine reductase 1 (PTR1). (9) PTR1 is responsible for the salvage of pterins in parasitic trypanosomatids and has overlapping activity with DHFR, providing a metabolic bypass to alleviate DHFR inhibition. (10-12) Under physiological conditions, PTR1 is responsible for only 10% of the reduction of folic acid required by the cell, but when classic antifolate drugs inhibit DHFR, the gene encoding the NADPH-dependent PTR1 is upregulated and PTR1 provides the reduced folates necessary for parasite survival. (13) Therefore, PTR1 is considered a promising target for the development of improved therapies. This protein is considered a validated target in T. brucei, even though a correlation between inhibition of T. brucei PTR1 (TbPTR1) and inhibition of parasite growth has not been demonstrated yet. The combination with MTX, a DHFR inhibitor, has been shown to improve the efficacy and the potency of PTR1 inhibitors. (14, 15)

In the drug discovery process, natural compounds represent a potential source of new leads. (16-19) Different natural compounds from plants including flavonoids have shown antitrypanosomatidic activity. (20, 21) Although flavonoids have been found to inhibit polyamine biosynthesis, the mechanism of action has not been fully elucidated. (21-24) Flavonoids exert pleiotropic effects, and different targets have been suggested to explain at least part of their antiparasitic activity. (25-27) Flavonoids have been proposed as PTR1 inhibitors only in silico. (28) Although some flavonoids are very effective in vitro, they lack notable in vivo activity due to their low bioavailability. (22) However, an efficient drug delivery strategy together with a clear definition of their molecular targets could lead to the exploitation of the antiparasitic potential of these natural products. (22)

The purpose of the present study was to identify new PTR1 inhibitors showing antitrypanosomal (T. brucei, T. cruzi) and antileishmanial (Leishmania infantum) activity by screening a library of natural products and then by using a medicinal chemistry approach to design, synthesize, and biologically evaluate a new library. Assessment of early ADME and toxicity properties provided selection criteria for further studies. MTX has previously been combined with PTR1 inhibitors to achieve synergistic/additive effects on T. brucei. (14) In our study, we evaluated the antiparasitic activity of the compounds both alone and in combination with MTX. Pharmacokinetic studies using BALB/c mice showed that one of our compounds (compound 2) was characterized by a short half-life, and therefore it was loaded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and solubilized with cyclodextrins, aiming to increase its stability and bioavailability.

Results and Discussion

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On-Target Screening of a Natural Product Library and Hit Identification

To identify potential new drugs for treating parasitic diseases, we investigated a library of natural products. Starting from a library of 98 compounds, a computational docking analysis against TbPTR1 together with consideration of structural diversity allowed the restriction of the library to 38 compounds that were tested for PTR1 inhibition. These 38 phytochemicals (NP-1–NP-38), consisting of twelve phenolic acids, five flavanones, nine flavones, six flavonols, two catechins, two triterpenes, and two anthraquinones, were investigated for in vitro inhibition of TbPTR1 and for growth inhibition of T. brucei. Due to their ability to bind similar folate-related metabolites, PTR1 inhibitors can also interact with other folate-dependent enzymes such as TS and DHFR both from parasite and human cells. Both human enzymes can be considered potential off-targets for PTR1 inhibitors, therefore all the compounds were assessed for their selectivity toward the human TS and human DHFR. The data obtained are depicted in Figure 1. All chemical structures and IC₅₀ values are reported in Supporting Information, Table S1.

Figure 1. Activity profile of the 38 phytochemicals screened against TbPTR1, hTS, and hDHFR and against the *T. brucei* parasite. The IC₅₀ is indicated by the color: dark-green, 0–30 μM; green, 31–90 μM; light-green, 90–150 μM; yellow, 151–250 μM; red, >250 μM; gray, not tested. *, Catechins; **, triterpenes; ***, anthraquinones.

With the exception of quercetin dehydrate (NP-30), all flavon-3-ol type aglycones showed a significant TbPTR1 inhibition, with 3-hydroxyflavone (NP-27, IC₅₀ = 12.8 μM) and isorhamnetin (NP-31, IC₅₀ = 13.9 μM) being the most potent, whereas the glycosylated flavonoid (NP-32) did not inhibit TbPTR1. Most phenolic acids showed no inhibitory activity toward TbPTR1, highlighting the importance of a condensed ring (chromen-4-one) for protein inhibition. The presence of two rings, one aromatic and the other one aliphatic (NP-8) or both aromatic (NP-10), slightly increased the inhibitory activity with respect to the other phenolic acid derivatives.

The simplest flavonol (NP-27, IC₅₀ = 12.8 μM) was 10 times more potent than the corresponding flavone (NP-18, IC₅₀ = 120.8 μM). This implies the importance of OH at position R3. Moreover, isorhamnetin (NP-31, IC₅₀ = 13.9 μM) was over 35 times more active against TbPTR1 than the corresponding flavone (chrysoeriol, NP-25, IC₅₀ > 490 μM). The insertion of a hydroxyl group at position R5 led to a decrease in the inhibitory activity. Indeed, the simplest flavone (NP-18, IC₅₀ = 120.8 μM) is more than 20-fold more active than primuletin (NP-19, IC₅₀ > 2450 μM), which has a hydroxyl group at position 5. On target screening of the NP series indicates that flavonols are the most promising compounds, with NP-31 being the most active and selective hit (selectivity index: SI hTS/TbPTR1 > 35; SI hDHFR/TbPTR1 = 36). Nevertheless, a clear structure–activity relationship (SAR) could not be established from this data set, and we could not reliably determine how the number and the pattern of hydroxyl/methoxy substituents influenced the activity.

The 38 compounds were also assessed for their inhibitory activity against T. brucei parasite (Figure 1, Supporting Information, Table S1). With the exception of NP-9, NP-10, NP-22, and NP-35, all the natural flavonoids screened showed antitrypanosomal activity with IC₅₀ lower than 30 μM. Figure 1 shows that there is no direct correlation between the antitrypanosomal activity of the NP compounds and their PTR1 inhibition effect.

X-ray Crystallographic Studies of the Natural Products

To understand the flavonoid interactions into the protein binding site, the 18 compounds showing IC₅₀ against TbPTR1 lower than 150 μM (compounds NP-8, NP-10, NP-12, NP-13, NP-18, NP-21, NP-22, NP-23, NP-24, NP-26, NP-27, NP-28, NP-29, NP-31, NP-35, NP-36, NP-37, and NP-38; Figure 1) were selected for structural analysis. We obtained crystal structures of the TbPTR1–NADPH/NADP⁺ complexes for two moderately active natural products (NP-29 (datiscetin) IC₅₀TbPTR1= 76.9 μM and NP-13 (hesperetin) IC₅₀ = 104.2 μM) only. These compounds show inhibitory activity against TbPTR1 and selectivity against at least one of the human proteins (TS and DHFR; Figure 1). Statistics for data collection and refinement are reported in the Supporting Information, Tables S2 and S3. In both cases, the crystal asymmetric unit contains the functional unit of the enzyme, the TbPTR1 tetramer. The tertiary structure of the TbPTR1 subunits is typical of the short-chain dehydrogenases/reductases (SDR) superfamily and shows a single α/β domain consisting of a seven-stranded parallel β-sheet sandwiched between two sets of α-helices. (29) The TbPTR1 active site is mainly formed by a single chain, with one end blocked by the C-terminus of the partner subunit. In this L-shaped depression, the cofactor binds in an extended conformation entrapped by a network of highly conserved hydrogen bonds. (29) The substrate-binding loop (residues 207–215) interacts with both the cofactor and the substrate. Two surface-exposed parts of the sequence, residues 104–112 and 143–151, are usually poorly visible in TbPTR1 crystal structures and they were not included in our models. The overall structure of each subunit of the TbPTR1 complexes is the same as indicated by the small root-mean-square deviations (RMSDs) after Cα superimposition of the four subunits in each tetramer (0.16–0.26 Å for NP-13 and 0.11–0.41 Å for NP-29). The binding of the cofactor is essential to create both the catalytic site and the substrate-binding pocket, indicating an ordered sequential reaction mechanism with NADPH binding before the substrate. (30) The pterin moiety of substrates and of pterin-like inhibitors binds in a π-sandwich between the nicotinamide ring of NADPH/NADP⁺ and the aromatic side chain of Phe97. (31) Pterin also interacts with the NADPH/NADP⁺β-phosphate, and this interaction is strongly conserved in pterin-like inhibitors. (31)

TbPTR1-NADPH/NADP⁺-NP-29

The chromen-4-one core of NP-29 binds in the biopterin binding pocket as described above. The ligand is supported by a network of H-bonds involving the cofactor and the surrounding residues in the active site cavity (see Figure 2A). The hydroxyl group at position 7 on the chromen-4-one of NP-29 (the NP-29 atom numbering is given in the inset in Figure 2A) donates a H-bond to the NADPH/NADP⁺β-phosphate and accepts a H-bond from an amino group on Arg14. The hydroxyl group at position 5 accepts a H-bond from NADPH/NADP⁺ ribose OH_2′ and donates a H-bond to the hydroxyl group of Tyr174, which donates a H-bond to the inhibitor carbonyl oxygen at position 4. Furthermore, the carbonyl oxygen and the hydroxyl group at position 3 of NP-29 are both H-bonded to a water molecule linked by a further H-bond to the side chain of Asp161. An additional water molecule, which is highly conserved throughout all TbPTR1 structures, mediates the interaction between the position 3 hydroxyl group of NP-29 and the backbone carbonyl oxygen of Gly205 (not shown). The o-phenol moiety of NP-29 is located in a hydrophobic pocket of the TbPTR1 active site cavity, surrounded by the side chains of Val206, Leu209, Pro210, Met213, and Trp221. The 2′ hydroxyl group on this ring is close (∼4 Å) to the carbonyl oxygen of Gly205 and forms an intramolecular H-bond (2.4 Å) with the hydroxyl group at position 3 of the chromen-4-one moiety.

Figure 2. Crystal structures of TbPTR1 (gray cartoon, interacting residues in sticks) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms) and four inhibitors (in sticks) (A) **NP-29** (cyan), (B) **NP-13** (orange), (C) compound 2 (lilac), and (D) compound 7 (yellow). Hydrogen bond interactions (dashed lines) in the active site are shown. The 2F_o – F_c electron density maps corresponding to the inhibitors (dark-blue wire) and NADPH/NADP⁺ (light-blue wire), contoured at the 1σ level are shown. The chemical structures and atom names of the ligands are specified in the insets.

TbPTR1-NADPH/NADP⁺-NP-13

The chromen-4-one core of NP-13 also binds in the biopterin binding pocket, but it is rotated by about 180° with respect to its orientation in the TbPTR1–NP-29 complex (Figure 2B). The ether oxygen at position 1 on the chromen-4-one moiety of NP-13 points toward the side chains of Asp161 and Tyr174. The hydroxyl group at position 7 of the NP-13 chromen-4-one is within H-bonding distance from the NADPH/NADP⁺ ribose OH_2′ and the side chain of Ser95. The NP-13 hydroxyl group at position 5 accepts a H-bond from an amino group of Arg14 and donates a H-bond to the backbone carbonyl oxygen of Leu208. The o-methoxy-phenol (o-guaiacol) ring in position 2 on the chromen-4-one makes a T-shaped stacking interaction with the aromatic side chain of Trp221. Furthermore, the 3′ phenolic moiety is within H-bonding distance from the Asp161 side chain and forms two water-mediated interactions with the backbone nitrogen of Cys168 and the amide nitrogen of Asn175 (not shown). The methoxy oxygen at position 4′ interacts with the sulfur of the modified Cys168 (S-oxy-cysteine), whereas the terminal methyl makes van der Waals contacts with the side chains of Trp221 and His267 of the partner subunit.

In summary, structural comparison of the two TbPTR1 complexes reveals two opposite orientations of the natural flavonoid inhibitors NP-13 and NP-29 in the biopterin binding pocket in which the chromen-4-one substituents interact with different residues surrounding the cavity. A double binding mode was previously observed for pteridine-like inhibitors and MTX. (15)

Flavonoid Library Design and Synthesis

The crystal structures showed that flavonoids occupy the biopterin binding site of TbPTR1, suggesting that the flavonol moiety could provide a new scaffold for the development of PTR1 inhibitors. The flavonoids selected as starting points for the development of compounds to treat trypanosomatidic diseases are depicted in Table 1. Even though the two crystal structures showed that the hydroxyl group at position 5 could establish H-bonds with the protein and the cofactor, the inhibitory data on natural flavonoids (Figure 1) suggested that a hydroxyl group in this position leads to a decrease in the inhibitory activity against TbPTR1. Therefore, we decided to synthesize compounds with hydroxyl substituents at positions 6 or 7 of the chromenone A-ring because, according to the crystal structures, these should be able to establish H-bonds with the NADPH/NADP⁺ ribose and the side chain of Ser95 or with the NADPH/NADP⁺β-phosphate and Arg14 (Figure 3). Moreover, we performed modifications at positions 3′, 4′, and 5′ on ring B to explore the SAR. As shown in Figure 3, hydroxyl groups in positions 3′, 4′, and 5′ could form a H-bonding interaction with the Asp161 side chain and two water-mediated interactions with the backbone nitrogen of Cys168 and the amide nitrogen of Asn175. Many natural products have both hydroxyl and methoxy groups, thus we synthesized and biologically evaluated eight hydroxylated (1–8) and eight methoxylated (9–16) compounds aiming to investigate how the number and the pattern of hydroxyl/methoxy substituents influenced the activity.

Table 1. Chemical Structures and IC₅₀ Values for Selected Compounds from the Screened Natural Products Library

compd	R₅	R₇	R_2′	R_3′	R_4′	IC₅₀TbPTR1 (μM)	IC₅₀T. brucei (μM)
NP-27	H	H	H	H	H	12.8	9.1
NP-28	OH	OH	H	H	OH	28.0	9.0
NP-29	OH	OH	OH	H	H	76.9	27.0
NP-31	OH	OH	H	OCH₃	OH	13.9	11.2

Figure 3. Design of the synthetic library (left). NP-13 (in yellow) into TbPTR1 (in gray). The amino acids involved in the interactions of the designed compounds are shown in different colors. For clarity reasons, two residues involved in the interactions are not shown (Cys168 and Asn175). Superimposition of the four crystal structures of TbPTR1 (right) (chain A gray ribbon; chain D pink ribbon; relevant active site residues as light-blue sticks) in complex with NADPH/NADP⁺ (sticks, black carbon atoms) and the inhibitors (stick) **NP-13** (orange), **NP-29** (cyan), compound 2 (lilac), and compound 7 (yellow). The three different binding modes adopted by the ligands can be appreciated as well as the movement of Trp221 (yellow sticks) upon binding of compound 7.

The synthesis of compounds 1–16 is depicted in Scheme 1. All the compounds have been already reported in literature, but as far as we know, they have never been proposed as PTR1 inhibitors. The intermediate chalcones (17–24) were synthesized by Claisen–Schmidt condensation using substituted acetophenones and benzaldehydes in the presence of NaOH as the base in ethanol. The reaction was carried out as previously reported in literature for similar compounds. (26) Afterward, the chalcones (17–24) were converted into the corresponding methoxylated flavonols (9–16), using the Flynn–Algar–Oyamada method for epoxidation and subsequent intramolecular cyclization of the open-chain structure. (25) The reaction was performed with hydrogen peroxide in aqueous base (1 M NaOH). Cleavage of methoxy protecting groups with boron tribromide gave the hydroxylated flavonols 1–8 in high yield. All synthesized compounds were characterized by ¹H NMR, ¹³C NMR, and mass analysis. The obtained NMR and mass data are reported in the Supporting Information (pp 33–37) and were compared with those available in literature.

Scheme 1. Synthesis of the Flavonols 1–16^a

Target Compound Profile

In our study, a panel of assays was performed and activity/toxicity data were considered during the hit selection/prioritization. (32) Together with the potency data (measured as IC₅₀, i.e., the compound concentration producing 50% reduction of targeted enzyme activity and parasite cell growth), we considered the selectivity index (IC₅₀ toward the parasite compared with compound cytotoxicity IC₅₀ on host cells) and early toxicity properties. We have assessed the entire profile of the 16 synthesized compounds, and the whole data panel has been evaluated before selecting compounds for pharmacokinetic studies.

Testing of Synthetic Flavonols against PTR1 Enzymes

All flavonols were investigated for their activity toward T. brucei (TbPTR1) and L. major PTR1 (LmPTR1). The results are shown in Figure 4 and Supporting Information, Table S4. All hydroxylated compounds except compound 5 showed significant inhibitory activity against LmPTR1 and TbPTR1 at 50 μM concentration, with compound 2 being the most potent. Methylation resulted in almost complete loss of inhibitory activity. The most noteworthy examples are represented by two pairs: compounds 10 and 2 (4% and 96% inhibition of TbPTR1 at 50 μM, respectively) and compounds 12 and 4 (no inhibition of TbPTR1 at 50 μM and 85% inhibition at 50 μM, respectively). The introduction of a hydroxyl group on ring A in compound 2 led to an increase of the inhibitory potency against both enzymes of almost 3-fold with respect to compound 1, which bears an unsubstituted ring (34% inhibition of TbPTR1 at 50 μM). To explain a complete SAR, X-ray crystallographic studies and docking analyses were carried out. The compounds were evaluated also toward TcPTR2, and none of the compounds significantly inhibited TcPTR2 activity (data not shown).

Figure 4. Inhibitory activity against TbPTR1 (in gray) and LmPTR1 (in black). The control compound was pyrimethamine, a PTR1 inhibitor (100% inhibition at 50 μM against both PTR1 enzymes).

Crystal Structures of Synthetic Flavonoid–TbPTR1 Complexes

Compounds 2 and 7, which show inhibitory activity against TbPTR1, were submitted to X-ray crystallographic characterization aimed at comparing their binding poses in the PTR1 active site with those of the natural flavonoids and thus gaining further information for compound optimization.

TbPTR1-NADPH/NADP⁺–2

The structure of TbPTR1 in complex with NADPH/NADP⁺ and compound 2 was determined at 1.38 Å resolution. Ligand placement and key interactions within the active site cavity are reported in Figure 2C. Compound 2, like NP-13 and NP-29, binds in the substrate binding pocket, with the chromen-4-one in a π-sandwich interaction between the nicotinamide ring of NADPH/NADP⁺ and the aromatic side chain of Phe97. The chromen-4-one core of compound 2 adopts the binding mode observed for NP-13, with the oxygen atom at position 1 directed toward the side chain of Asp161. The R6 hydroxyl group on the chromen-4-one is within H-bonding distance from the hydroxyl group of Ser95 and the NADPH/NADP⁺β-phosphate oxygen. The carbonyl oxygen at position 4 completes the interactions made by the chromen-4-one by receiving two H-bonds from an amino group of Arg14 and a water molecule that is H-bonded to the NADPH/NADP⁺β-phosphate O2. These interactions, together with the stacking of the chromen-4-one described above, bring its 3 position hydroxyl group within H-bonding distance from the backbone carbonyl oxygen of Leu208. The phenyl ring in position 2 on the chromen-4-one establishes a T-shaped stacking interaction with the aromatic side chain of Trp221 and forms van der Waals interactions with the side chains of Val206 and Leu209 (not shown). The meta-hydroxyl group at the 3′ position on the phenyl moiety of 2 is only involved in a water mediated H-bond network linking it to the backbone carbonyl of Gly205 and the side chain of Asp161 (not shown).

TbPTR1-NADPH/NADP⁺–7

The structure of TbPTR1 in complex with NADPH/NADP⁺ and compound 7 has been determined at 1.76 Å resolution. Ligand placement and key interactions within the active site cavity are shown in Figure 2D, where it can be observed that compound 7 adopts exactly the same pose as compound 2, making the same interactions with the enzyme and cofactor. The only difference consists in the steric repulsion of the additional hydroxyl group at position 4′ that forces the side chain of Trp221 to move away by about 1.4 Å. The 4′ hydroxyl group is not involved in H-bonds but is solvent exposed and presumably can form a H-bond with water.

The superposition of the four crystal structures is shown in Figure 3. The orientation of compounds 2 and 7 in the TbPTR1 cavity is the same as for NP-13, with the inhibitor bicyclic core directed toward the opposite side of the cavity with respect to NP-29. Due to the larger steric hindrance of the substituents on the 2-o-methoxy-phenolring of NP-13, a little rearrangement of the whole molecule occurs with a rotation of about 35° of the bicyclic core with respect to 2 and 7 that allows the side chain of Trp221 to remain in the position observed in the TbPTR1–2 complex (Figure 2C). The rotation of NP-13 allows direct H-bonding between the 3′ hydroxyl group of NP-13 and Asp161, while the interaction of the corresponding group of 2 and 7 is mediated by a water molecule. The displacement of the Trp221 side chain occurring upon binding of compound 7 can be appreciated (Trp221 as yellow sticks in Figure 3, right).

Figure 5. Comparison of the effects of hydroxyl substituents at the R6 (left) and the R7 (right) positions of ring A on the binding of the synthetic flavonoids to TbPTR1. Superimposition of constraint docking poses for compound 2 (in sticks, lilac carbons) and compound 5 (in sticks, pale-green carbons) (left) and compound 3 (in sticks, orange carbons) and compound 6 (in sticks, brown carbons) (right) in TbPTR1 (in cartoon with interacting residues in sticks with gray carbons) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.

A puzzling aspect of all four crystal structures is that the different inhibitors have been systematically observed bound to three of the four TbPTR1 subunits, whereas the NADPH/NADP⁺ cofactor is found bound to all subunits in three instances out of four, although with lower occupancy in those subunits where the inhibitor is absent. The absence of the inhibitor is correlated with larger disorder in the substrate binding loop that brings Trp221 into the active site cavity. However, this loop always shows temperature factors higher than average, even in the subunits where both cofactor and inhibitor are bound. The disorder of this region is independent of the crystal packing because, when the loop is disordered (e.g., in subunit C in TbPTR1–NP-29), it approaches the symmetry-related subunits in the same way as the ordered loop (e.g., in subunit A in TbPTR1–NP-29) located on the opposite face of the TbPTR1 tetramer. Confirming this observation, the other two ordered loops (e.g., in subunits B and D in TbPTR1–NP-29) do not make close intermolecular contacts. We can conclude that stabilization due to the π-stacking between the R2-aromatic ring of the inhibitor and the side chain of Trp221 is critical for the ordering of the substrate binding loop.

Computational Docking of Synthetic Compounds to TbPTR1 and LmPTR1 and SAR Analysis

Docking studies were conducted to further expand the SAR to all the hydroxylation/methoxylation patterns in the synthesized compounds. In addition, computational docking allowed the evaluation of possible binding modes and a corresponding SAR for LmPTR1, for which no crystal structure with these compounds could be solved.

Like the crystallographic studies, computational docking showed multiple possible binding modes for the chromen-4-one system, which roughly group into three classes: (1) a typical substrate-like binding mode involving H-bonding with the NADPH/NADP⁺ cofactor and Ser95 of TbPTR1 (or Ser111 of LmPTR1), as observed in the complexes of compounds 2, 7, and NP-13; (2) an alternative binding mode with chromen-4-one flipped by roughly 180° as observed in the complex with NP-29; and (3) an inverse binding mode solely observed in docking studies, with ring B rather than the chromen-4-one in a stacking orientation between the NADPH/NADP⁺ nicotinamide and Phe97 of TbPTR1 (or Phe113 of LmPTR1). While three of the four crystal structures feature binding mode (1), a general dependence of the binding mode on the substituent pattern is notable both from crystallographic and docking studies. In docking, many of the observed binding modes were found to have highly similar contacts and H-bonding partners, thus creating a favorable entropic contribution to the binding of this compound class (see also Supporting Information, Tables S5 and S6). However, due to their large number of H-bond donors/acceptors, flavonoids can adopt additional binding modes with different, often water mediated, interactions. We therefore used a ligand constraint docking approach to favor arrangements close to the crystal complexes to assess the potential of other synthetic derivatives to bind in similar orientations.

The crystal structures and docking indicate that hydroxylation patterns on ring A influence the orientation of chromen-4-one in the pocket. Precisely, compounds hydroxylated at position R6 show different H-bonding partners (mostly NADPH/NADP⁺ phosphate and Ser95/Ser111 of TbPTR1/LmPTR1, respectively) compared with those hydroxylated at R7 (mostly NADPH/NADP⁺ ribose and Tyr174/Tyr194 or TbPTR1/LmPTR1, respectively), see Figure 5. This difference also affected the compound activity (compare R6-hydroxylated compound 2 (96% TbPTR1 inhibition at 50 μM) and the corresponding R7-hydroxylated compound 3 (47% TbPTR1 inhibition at 50 μM)). While the geometry of compound 2 fits ideally in the TbPTR1 binding pocket, the slight reorientation observed for compound 3 leads to a loss of H-bonds and stabilizing hydrophobic contacts, halving the compound activity. Thus, hydroxylation of ring A can fine-tune the arrangement of the chromen-4-one system within the pocket and critically influence the interaction pattern of rings B and C.

Methoxylations, in contrast to hydroxylations, were found to lead to activity loss. For example, the inhibitory activity against TbPTR1 and LmPTR1 drops substantially when comparing compound 2 with the corresponding methoxylated compound 10 (96% and 4% TbPTR1 inhibition at 50 μM; 86% and no LmPTR1 inhibition, respectively). Methoxylations on the A-ring cause a displacement of the chromen-4-one from the primary stacking geometry to accommodate the bulkier methoxy group in close proximity to the NADPH/NADP⁺ cofactor and Ser95 of TbPTR1 (Ser111 of LmPTR1), see Figure 6, left. Notably, this adverse effect was also apparent in the comparison of compounds 1 and 9, bearing no substitution on ring A but a hydroxylation or methoxylation on R3′ of the B-ring. Whereas the activity of compound 9 is negligible, compound 1 shows about 35% inhibition against both TbPTR1 and LmPTR1 (at 50 μM). A H-bond donor functionality on R3′ has an important stabilizing effect, as it can interact with a water molecule bridging Asp161 and Gly205 of TbPTR1 (see Figure 6, right) or Asp181 and Gly225 of LmPTR1. As this water, according to our WatCH analysis, is almost 100% conserved in both TbPTR1 and LmPTR1, it provides a clear advantage for R3′ hydroxylated compounds. This is further supported by the crystal structures of TbPTR1 with compounds 2 and 7, where the R3′ hydroxylation is in H-bonding distance to this water site. In agreement with this observation, compound 2 with the m-hydroxyphenyl was over 30 times more active than compound 5 with a p-hydroxyphenyl ring (96% and 3% TbPTR1 inhibition at 50 μM, respectively). In contrast to a meta-hydroxylation, a para-hydroxylation cannot interact with the structural water, see Figure 5. Again, this effect was sensitive to the hydroxylation pattern of ring A, as can be observed by comparing compounds 3 (R3′ and R7 hydroxylation, 47% TbPTR1 inhibition at 50 μM) and compound 6 (R4′ and R7 hydroxylation, 33% TbPTR1 inhibition at 50 μM), see Figure 5. While p-substituted compounds generally show a drop in inhibitory activity compared to their respective m-substituted counterparts, the effect is weaker for the second pair with the R7 hydroxylation on ring A, possibly because R7 substitutions lead to a slight rotation of the stacking chromen-4-one, directing the p-hydroxyl toward a more solvent-exposed region of the pocket. In conclusion, the effects of individual hydroxyl substituents are not purely local and do not show a completely additive effect; rather, the combinations of ring A and B hydroxylations will determine the final binding mode.

Figure 6. Comparison of the effects of hydroxyl and methoxy substituents at the R6 (left) and the R3′ (left and right) positions on the binding of the synthetic flavonoids to TbPTR1. Constraint docking poses are shown for compound 2 (in sticks, lilac carbons), compound 10 (in sticks, dark-green carbons) (left), compound 9 (in sticks, pale-cyan carbons), and compound 1 (in sticks, magenta carbons) (right) in TbPTR1 (in cartoon with interacting residues in sticks with gray carbons) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.

On the basis of the binding modes observed for TbPTR1, we evaluated possible binding orientations to LmPTR1 by docking. Notably, all compounds with R4′ hydroxylation (compounds 5 and 6), compounds with R3′ and R4′ hydroxylations (compounds 7 and 8) and one compound with R3′ hydroxylation combined with an R7 substitution on ring A (compound 3) were found to be slightly more active toward LmPTR1 than TbPTR1. While the depth of the biopterin binding pocket is well conserved and the interactions for chromen-4-one are practically identical in both proteins, there are notable differences in the opening of the pocket that can explain the observed activity differences (see sequence alignment of interaction partners in Supporting Information, Figure S2). While in TbPTR1, R3′ hydroxylations on the B-ring mainly come into contact with the conserved water molecule and Cys168 (see Figure 5) and R4′ hydroxylated compounds can hardly form direct H-bonds with the receptor, LmPTR1 generally has a more polar opening part of the pocket. Three major differences are crucially important: His241, Tyr283, and Arg287 from the neighboring subunit in LmPTR1, correspond to Trp221, Leu263, and His267 from the neighboring subunit in TbPTR1, thereby providing several additional polar contact points. As the side chain of Arg287 in LmPTR1 is much bigger than that of His267 in TbPTR1, arginine is much more accessible to the ligands than histidine. Furthermore, as observed in crystal structures of TbPTR1, Trp221 is rather motile and different rotamers lead to an open or a rather closed form of the pocket, thereby potentially blocking ligand access to His267. While Trp221 of TbPTR1 did not provide a H-bonding contact to any of the studied compounds, induced fit docking studies of the compounds to LmPTR1 showed movement of His241 to allow additional H-bonding interactions in the most stable structure, as also shown in Figure 7 (see also Supporting Information, Table S5). Overall, π–π stacking and hydrophobic contacts are clearly the main stabilizing factors for flavonoids in TbPTR1, whereas a tendency toward a higher number of H-bond donor/acceptor contacts may stabilize hydroxylations in both the meta- and para-positions of ring B slightly better in LmPTR1.

Figure 7. Superimposition of the crystal structure of LmPTR1 (PDB ID 1E92) in cartoon representation and interacting residues in sticks representation. Chains A and D (containing Arg287) are colored in pale-pink and magenta, respectively) and the best predicted receptor conformation obtained in the induced-fit docking study starting from this crystal structure (His241 in H-bonding contact to compound 7) in complex with NADPH/NADP⁺ (in sticks, black carbons) and compound 7 (in sticks, yellow carbons). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.

While addition of a para-hydroxylation for the R7 substituted ring A did not lead to a significant change in activity against TbPTR1 (compound 3, 47%; compound 8, 53% TbPTR1 inhibition at 50 μM), compound 4 with hydroxylations at R3′, R4′, and R5′ yielded a clear activity increase (85% TbPTR1 inhibition at 50 μM). Considering the lack of direct polar contact points in TbPTR1, this is surprising and there may be two possible explanations: (1) bridging water molecules may enable additional contacts for the second meta-hydroxylation, e.g., with Trp221, or (2) the compound may adopt an inverted binding mode, placing the pyrogallol moiety in a stacking orientation with Phe97 and the cofactor, where it could make strong H-bonds and thereby keep the more hydrophobic chromen-4-one in the hydrophobic TbPTR1 subpocket, where it is able to form additional hydrophobic contacts.

Testing of Synthetic Flavonols against TbDHFR, LmDHFR, hDHFR, and hTS

The compounds showing inhibitory activity against PTR1 (1–8) were evaluated for their activity against TbDHFR, LmDHFR, and hDHFR. The results are reported in the Supporting Information, Table S7. With the exception of compounds 1 and 2, all the tested compounds had inhibitory activities against TbDHFR and LmDHFR below 25% at 50 μM. Compounds 1 and 2 slightly inhibit, respectively, TbDHFR and LmDHFR (42–43% at 50 μM). Therefore, we can conclude that compounds 1–8 have low inhibitory activity against the parasitic DHFRs. With the exception of compound 2, all the compounds had inhibitory activities against hDHFR below 30% at 50 μM. The IC₅₀ of compound 2 against hDHFR is 50 μM and against TbPTR1 is 4.3 μM, thus compound 2 has a selectivity index higher than 10. None of the tested compounds inhibited hTS at 50 μM (data not shown).

Early Toxicity Studies

To characterize the compounds properties and to define their toxicological profile, we used an assay panel typical of those employed in drug discovery projects for hit/lead selection. (32) Our panel includes hERG, five cytochrome P450s, (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), Aurora B kinase, A549 (human lung adenocarcinoma epithelial) and WI-38 (fetal lung fibroblasts) cell lines and mitochondrial toxicity. The results of the screening are reported in Figure 8 as a traffic light code (from red through yellow to green for increasing biological effect and decreasing toxicity of the compounds) and in Supporting Information, Table S8. An ideal lead compound should have all parameters colored in green. In detail, the percentage of mitochondrial toxicity and of inhibition toward hERG, CYP isoforms, and Aurora B kinase should be below 30%, while the percentage of A549/WI-38 cells growth, measuring cytotoxicity, should be above 70%. A few compounds showed a percentage of inhibition toward hERG > 10% at 10 μM. Apart from compounds 4 and 8, all the compounds inhibited at least one isoform of cytochrome P450 (mostly CYP1A2), while only two compounds (4 and 8) exhibited a significant inhibitory activity against Aurora B kinase at 10 μM. None of the compounds was either cytotoxic or showed mitochondrial toxicity.

Figure 8. Early toxicity properties combined with inhibitory activity against *T. brucei*. The data are reported as a traffic light system. An ideal compound (I. comp.) should have all the parameters green. The cells are colored in green when the percentage of inhibition of *T. brucei* and the percentage of A549 and W1–38 cell growth is between 60 and 100, while the percentage of inhibition of CYP isoforms, hERG, Aurora B kinase and mitochondrial toxicity is between 0 and 30. Cells are colored in red when data indicates toxicity or inactivity. Yellow stands for a borderline value (30–60%): moderately active or slightly toxic compound. Gray: not tested.

Testing of Synthetic Compounds on Cultured Parasites

With the exception of compound 8, all the molecules were tested at 10 μM against T. brucei bloodstream form, intracellular T. cruzi, and L. infantum intracellular amastigotes (Figure 9). Almost all tested compounds were active against T. brucei, while only two compounds, 4 and 12, were slightly active against L. infantum, showing inhibitory activities of 10 and 35%, respectively. Twelve compounds (1, 2, 5–12, 14–16) presented EC₅₀ values against T. brucei between 1 and 8 μM (Table 2), while compounds 3, 4, and 13 showed EC₅₀ values of 12.29, 18.04, and 20.12 μM, respectively. Only the methoxylated compounds 10, 11, 14, and 16 showed antiparasitic activity toward T. cruzi trypomastigotes similar to that of nifurtimox (NFX), the reference drug for the treatment of Chagas’ disease. Compound 10 is the most active (EC₅₀ = 4.5 μM, NFX EC₅₀ = 1.6 μM).

Figure 9. Antiparasitic activity of the synthesized compounds against *Trypanosoma brucei* (in gray), *Trypanosoma cruzi* (in green), and *Leishmania infantum* (in black) at 10 μM. The reference compounds were pentamidine (IC₅₀ = 1.55 ± 0.24 nM) for *T. brucei*, miltefosine (IC₅₀ = 2.65 ± 0.4 μM) for *L. infantum*, and nifurtimox (IC₅₀ = 2.2 ± 0.4 μM) for *T. cruzi*.

Table 2. EC₅₀ against T. brucei, NOAEL, and Selectivity Index of the Synthesized Compounds^a

compd	EC₅₀T. brucei (μM) ± SD	CC₅₀ ± SD or NOAEL	selectivity index (CC₅₀/EC₅₀)
1	5.18 ± 1.10	20	4
2	7.56 ± 0.51	53 ± 2	7
3	12.29 ± 2.82	80 ± 2	6
4	18.04 ± 0.50	100	5
5	2.32 ± 0.42	20	7
6	4.29 ± 0.71	20	4
7	1.36 ± 0.57	10	6
9	2.32 ± 1.01	10	4
10	1.43 ± 0.42	10	7
11	1.14 ± 0.24	10	8
12	1.17 ± 1.07	20	17
13	20.12 ± 2.27	20	1
14	1.10 ± 0.47	10	9
15	2.02 ± 0.51	10	5
16	2.99 ± 1.86	25	8
pentamidine	0.00155 ± 0.00024	10	6440

EC₅₀ and NOAEL represent the arithmetic average of at least two independent determinations done in triplicate.

The series was assessed for cytoxicity on THP1 macrophage-like cells to evaluate the NOAEL (no observed adverse effect level). The reference compound was pentamidine with a NOAEL of 10 μM and a selectivity index of 6440. The selectivity indexes of our compounds, given by the ratio between the EC₅₀ toward T. brucei and CC₅₀ toward THP1, were lower than 10 with the exception of compound 12 (SI = 17) (Table 2). The selectivity index acceptable for a compound to be considered non toxic (33) is at least 10, thus our compounds showed slight toxicity.

Combination Studies and Evaluation of Synergy

All compounds were evaluated in combination with MTX independently of their PTR1 inhibitory activity against the recombinant protein. We aimed to assess the potential gain in potency and the reduction in toxicity through the combination of our compounds with MTX, a well-known DHFR inhibitor. In trypanosomatids, MTX is expected to exert a synergistic behavior in combination with PTR1 inhibitors. Moreover, MTX can show a synergistic effect in combination with non-PTR1 inhibitors through a multitarget inhibition. Preliminary combination experiments in T. brucei were conducted by fixing MTX at 4 μM (MTX average EC₃₀ against T. brucei) and varying the concentration of the selected compounds between 1.25 and 20 μM (Supporting Information, Table S10). The synergy coefficients of all the compounds tested at the different concentrations (1.25, 2.5, 5, 10, 20 μM) are reported in Supporting Information, Table S11. Eight compounds presented a synergistic effect (synergy coefficient >1) when combined with MTX. The antiparasitic activity against T. brucei of the eight compounds both alone and in combination with MTX is shown in Figure 10. Compounds 5, 6, and 13 consistently presented the highest synergy coefficients (maximum synergy coefficient >1.5). Compound 13 was the most synergic with a calculated maximum synergy coefficient of 3.1 at 1.25 μM (Supporting Information, Table S11). Since it showed the highest synergy coefficient together with a safe toxicological profile (Figure 8, the only poor (red) parameter is for CYP1A2 (72% inhibition at 10 μM)), it was selected for further synergistic combination studies.

Figure 10. Trypanocidal activity of eight synthesized compounds alone and in combination with MTX. The compounds were tested at three different concentrations: 5 μM (in green), 2.5 μM (in light-pink), 1.25 μM (in light-blue). Antiparasitic activity of MTX is shown in dark-red.

To better evaluate the gain in potency of the combination MTX–13, we employed the software Compusyn and a constant ratio between the concentrations of the two compounds chosen from their EC₅₀ values (EC₅₀13 = 20 μM; EC₅₀ MTX = 15; constant ratio = 1.3). (34) This enabled us to quantify synergism between the two compounds (combination index, CI) and to determine the dose reduction (dose reduction index, DRI) needed to observe the studied effect in the combination compared with the dose of each drug alone. The combination of MTX and compound 13 at the EC₅₀ concentration had a CI of 0.174 ± 0.047. When CI is lower than 1, the combination can be considered synergistic, therefore the observed CI shows strong synergism and confirms the initial data (Supporting Information, Table S10). At higher EC values, CI becomes lower, indicating very strong synergism (Table 3). The EC₅₀ determined for the combination showing CI of 0.147 was 3.15 ± 0.33 μM. This enabled an average DRI at the EC₅₀ of 14.6 ± 3.7 for MTX and of 11.7 ± 2.7 for compound 13 (IC₅₀ MTX = 1.3 μM and IC₅₀13 = 1.7 μM) (Supporting Information, Figure S3C). At the EC₉₀ of the mixture, the predicted DRI is over 100-fold for both compounds when compared with the EC₅₀ of the compounds alone. The isobologram is reported in Supporting Information, Figure S3. The toxicity of the MTX–13 mixture on THP1 was determined at three different concentrations (12.5, 25, 50 μM), and no synergistic toxicity on human cells was observed (Supporting Information, Figure S4 and Table S12). Therefore, the identified combination MTX–13 is selective for the inhibition of the parasite growth, while there is no synergy in the toxicity toward the host cells. These data together with the predicted DRI indicate that the combination improves the selectivity indexes for both MTX and compound 13.

Table 3. Summary Report of Computer-Simulated CI and DRI Values for MTX and Compound 13 Combinations at 50%, 75%, 90%, and 95% Inhibition of T. brucei Growth^a

		CI values at the reported EC				DRI values at the reported EC
drug combination	combination ratio	EC₅₀	EC₇₅	EC₉₀	EC₉₅	EC₅₀	EC₇₅	EC₉₀	EC₉₅
MTX + 13	(2:1.5)	0.174 ± 0.047	0.075 ± 0.036	0.034 ± 0.023	0.020 ± 0.016	14.58 ± 3.71^b	41.95 ± 10.52^b	127.36 ± 60.20^b	278.17 ± 172.13^b
						11.72 ± 2.79^b	27.45 ± 6.90^b	65.56 ± 16.42^b	119.72 ± 29.15^b

Data reported are the average of three independent experiments. Data analysis was carried out using the CompuSyn software. (34).

Values on top are for the MTX and on the bottom are for compound 13

Pharmacokinetic Studies, in Vivo Assays, and Compound Delivery

Compound 2 was selected for pharmacokinetic studies because it was the most active compound against TbPTR1 (IC₅₀ = 4.3 μM) that showed activity against the parasite T. brucei (EC₅₀ = 7.6 μM) and it presented a safe profile, see Figure 8. The hERG IC₅₀ (99.3 μM) was more than 20-fold higher than the target IC₅₀. With the exception of the CYP1A2 IC₅₀ (6.1 μM), the IC₅₀ against the cytochrome isoforms was higher than 10 μM. The solubility of compound 2 was evaluated using UV–Vis spectroscopy. In vivo bioavailability and half-life of compound 2 were evaluated in BALB/c mice treated IV with 1 mg/kg based on its low solubility. The molecule showed a short half-life (t_1/2 = 7.6 min), and it was not detected in blood after 30 min (the plasma levels are shown in Supporting Information, Figure S4). With the aim of improving the plasma levels of compound 2, we used two different drug delivery systems: the molecule was encapsulated in PLGA nanoparticles and solubilized with hydroxypropyl-β-cyclodextrins. The encapsulation did not change the physical properties of the PLGA nanoparticles as reported in Supporting Information, Table S13. The unloaded and loaded NPs were characterized by dynamic light scattering (DLS) in terms of size, polydispersity index, and ζ potential. We evaluated the in vivo bioavailability of compound 2 encapsulated in nanoparticles in BALB/c mice (IV and per os administration) and formulated with cyclodextrins after per os administration to NMRI mice. From preliminary data, both formulations did not significantly increase the levels of compound 2 in plasma samples from mice (data not shown). However, the in vitro activity of compound 2 both in PLGA and in cyclodextrins solution was preserved when compared to the free compound while the toxicity on THP1 was diminished (Table 4).

Table 4. in Vitro Activity against T. brucei of Compound 2 in Three Different Formulations^a

preparation	T. brucei EC₅₀ (μM)	THP1 CC₅₀ ± SD or NOAEL
2	7.56 ± 0.51	53 ± 2
PLGA-2	5.70 ± 0.23	>100
cyclodextrin-2	3.27 ± 0.16	>100

EC₅₀ and NOAEL are the arithmetic mean of at least two independent determinations done in triplicate.

Conclusion

ARTICLE SECTIONS

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This is the first study in which we have confirmed the potential of flavonols as PTR1 inhibitors. Apart from compounds 4 and 8, none of the 16 synthesized flavonols have previously been reported in literature for their antiparasitic activity. (22) Twelve of these compounds show EC₅₀ values against T. brucei below 10 μM. We performed combination studies with MTX and compound 13, a non-PTR1 inhibitor, which was the best-performing compound. Although the reason for the synergy is unknown, compound 13 is an interesting compound for drug development due to the synergistic behavior with MTX and the low toxicity on host cells. Target identification studies could be carried out aiming to explain the mechanism of action. The synthesized compounds were also evaluated for the activity against T. cruzi trypomastigotes, and compound 10 turned out to have a potency comparable to that of nifurtimox, the drug currently used to treat Chagas’ disease (EC₅₀ compound 10 = 4.5 μM, NFX EC₅₀ = 1.6 μM). We carried out early in vitro toxicity assays, and overall the library showed a satisfactory profile. Combining the activity/toxicity data, we selected compound 2 for pharmacokinetic studies and we observed a very quick turnover of the molecule in BALB/c mice. Neither encapsulation in PLGA nanoparticles nor solubilization with cyclodextrins increased the plasma level of compound 2; however, both formulations allowed to maintain the in vitro activity of the compound and to reduce the toxicity. Our crystal structures and SAR analysis provide a basis for structure-based drug design aimed at identifying novel flavonol-like compounds with increased potency and selectivity toward PTR1 and able to overcome the problems of classical flavonols.

Experimental Section

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Preparation of the Natural Products Library

A library of 98 natural compounds, consisting of 8 flavanones, 19 flavones, 20 flavonols, 2 dihydroflavonols, 13 anthocyanins, 2 catechins, 2 isoflavones, 4 chalcones, 18 phenolic acids and derivatives, 1 aurone, 5 anthraquinones, 3 triterpenes, and 1 phloroglucinol was purchased from Extrasynthese (Genay, France) and Sigma-Aldrich-Fluka (Milan, Italy) for drug screening. The degree of purity of all these molecules was checked by HPLC. The HPLC analyses were performed on an Agilent Technologies (Waldbronn, Germany) modular model 1100 system, consisting of a vacuum degasser, a quaternary pump, an autosampler, a thermostated column compartment, and a diode array detector (UV/DAD). The chromatograms were recorded using an Agilent Chemstation for LC and LC-MS systems (Rev. B.01.03). An Ascentis C₁₈ column (250 mm × 4.6 mm I.D., 5 μm, Supelco, Bellefonte, PA, USA) was used, with a mobile phase composed of (A) water (H₂O) and (B) acetonitrile (ACN). The gradient elution was modified as follows: 0–3 min 25% B, 3–10 min from 25 to 30% B, from 10−40 min from 30 to 40% B, which was held for 5 min. The postrunning time was 5 min. The flow rate was 1.0 mL/min. The column temperature was set at 30 °C. The sample injection volume was 5 μL. The UV/DAD acquisitions were carried out in the range 190–600 nm, and chromatograms were acquired at 310, 330, 370, and 520 nm. All the compounds tested were found to be stable, and degradation products were not detected in the HPLC chromatograms. Supporting Information, Table S1 shows the purity (>95%) of the 38 natural compounds screened. The natural compounds were stored at low temperature (−80 °C), protected from light and humidity.

Computational Studies

Virtual Screening of the Natural Product Library

The library of 98 natural products was subjected to virtual screening using the GOLD software (version 5.1) (35, 36) following the protocol validated in previous studies on PTR1. (13) The compounds were docked into the binding sites of the targets, LmPTR1 (PDB ID 1E92) and TbPTR1 (PDB ID 3JQ9), and the off-targets, hDHFR (PDB ID 1U72) and hTS (PDB ID 1HVY). Ten docking poses were generated for each ligand in each target, and these were visually inspected and manually ranked, considering the Gold fitness score, hydrogen bonding, and π–π aromatic interactions. A search of BioAssay data in PubChem was done to filter out promiscuous compounds. The set of 38 compounds given in Supporting Information, Table S1 was selected for inhibition assays considering the above properties and structural diversity.

Docking of Synthetic Flavonoid Derivatives

The 3D structures of the compounds were created from SMILES strings and optimized with the OPLS_2005 force field using Maestro. (37) Ionization states and tautomers were generated at pH 7.0 ± 0.5 using Epik. (37) Up to eight stereoisomers and one low-energy ring conformation were generated per compound. Important structural water sites were identified using the WatCH clustering approach. (38) All available chains of the LmPTR1 crystal structures and 99 chains from TbPTR1 crystal structures (see Supporting Information, Tables S14 and S15 for a list of structures) were superimposed and their water sites were clustered with a distance criterion of 2.4 Å. Water sites with at least 50% conservation were considered conserved.

The PTR1 structures for docking consisted of chain A of the relevant crystal structure and a C-terminal tripeptide from the neighboring subunit pointing into the chain A active site (Val266 to Ala268 in TbPTR1 and Thr286 to Ala288 in LmPTR1). The side -chain oxygen atom on the modified Cys 168 in the crystal structure of TbPTR1 was removed. Crystallographic solvent molecules were removed and replaced by the conserved water molecules identified by WatCH clustering. The LmPTR1 structure (PDB ID 1E92) was aligned to the TbPTR1 template (PDB ID 5JCJ). PrepWizard was used to assign bond orders and add hydrogen atoms. (37) The N- and C-termini of chain A were capped with N-acetyl and N-methyl amide groups, respectively. The protonation state of the NADPH/NADP⁺ cofactor was computed at pH 7.0 ± 0.5. Protein protonation states were assigned by PROPKA (43) at pH 7.0. The H-bond network was optimized and all hydrogens were subjected to a restrained minimization. A 20 Å × 20 Å × 20 Å docking grid was centered on Phe97 of TbPTR1 or Phe113 of LmPTR1. The following hydroxyl groups were set rotatable: Ser95 and Tyr174 in TbPTR1 and Ser111, Thr184, Tyr191, Tyr194, Thr195, and Tyr283 in LmPTR1, as well as those of the NADPH/NADP⁺ ribose.

Docking was performed with and without ligand-based constraints using the Glide software. (39-42) For the ligand constraints, protein preparation was performed including compound 7 (substrate-like binding mode, PDB ID 5JCJ) or compound NP-29 (inhibitor-like binding mode, PDB ID 5JCX) or the highest scoring docking solution from unconstrained docking of compound 4 with ring B in a stacking orientation (alternate binding mode). The ligands were then separated from the structure to provide reference geometries. The van der Waals radii of the ligand atoms were scaled by 0.80, and a partial charge cutoff of 0.15 was used. XP (extra precision) docking and flexible ligand sampling were set. Nitrogen inversions and ring conformations were sampled, and biased sampling of torsions was performed for amides only and set to penalize a nonplanar conformation. Epik state penalties were added to the docking score, intramolecular H-bonds were rewarded, and the planarity of conjugated π groups was enhanced. A core pattern comparison with a tolerance of 3 Å was used for the ligand-based constraint. The core was defined as all non-hydrogen atoms of the chromen-4-one core ring system except the R3 hydroxyl group. Twenty poses per ligand were subjected to postdocking minimization with a threshold of 0.5 kcal/mol for rejecting a minimized pose. Up to 10 final docking solutions were reported. Unconstrained docking was performed analogously, omitting only the core pattern comparison. For LmPTR1, the core constraint docking was based on the binding poses in TbPTR1. In addition, induced-fit docking was carried out using the same settings except that the size of the docking grid was chosen automatically and a ligand and receptor van der Waals scaling of 0.5 was applied. Refinement was carried out with Prime (37) for residues within 5 Å of the ligand, followed by XP redocking into structures within 30 kcal/mol of the best structure and within the top 20 structures overall. Up to 20 final docking solutions were reported.

Synthesis

General

All commercial chemicals and solvents were reagent grade and were used without further purification. Reaction progress was monitored by TLC on precoated silica gel 60 F254 plates (Merck), and visualization was accomplished with UV light (254 nm). ¹H and ¹³C NMR spectra were recorded on a Bruker FT-NMR AVANCE 400. Chemical shifts are reported as δ values (ppm) referenced to residual solvent (CHCl₃ at δ 7.26 ppm, DMSO at δ 2.50 ppm, MeOD at δ 3.31 ppm); J values are given in Hz. When peak multiplicities are given, the following abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadened signal. Silica gel Merck (60–230 mesh) was used for column chromatography. All the reactions were monitored using TLC (Merck 890 F-254 silica gel). Mass spectra were obtained on a 6520 Accurate-Mass Q-TOF LC/MS and 6310A Ion Trap LC-MS(n). The detailed NMR and mass data of the synthesized compounds are reported in the Supporting Information (pp 33–37).

General Procedure for the Synthesis of (2E)-1-(2-Hydroxy-phenyl)-3-phenylprop-2-en-1-ones

An aqueous solution of NaOH (3 M, 1.6 mL) was added to a solution of aromatic ketone (1 mmol) and substituted benzaldehyde (1.2 equiv) in EtOH (1–2 mL). The reaction was stirred at room temperature overnight. The reaction mixture was cooled in an ice–water bath and acidified to pH 2 with concentrated HCl (37%). The solid formed was filtered, washed with ethanol, and then further purified by recrystallization from EtOH.

General Procedure for the Synthesis of Methylated Flavonols (Compounds 9–16)

An aqueous solution of H₂O₂ (30%, 250 μL) was added to an ice-cold suspension of the chalcone (1 mmol) in ethanol (5 mL) and 1 M NaOH (2 mL). The mixture was allowed to warm to room temperature and was stirred for 5–18 h. Then the reaction mixture was cooled in an ice bath, and distilled water (2–4 mL) was added. Concentrated HCl (37%) was added until pH 2, and the precipitate was filtered, washed with EtOH, and further purified by recrystallization from EtOH. When no precipitate occurred, the reaction mixture was extracted with dichloromethane and washed with water and brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. Column chromatography was utilized to purify the desired product.

General Procedure for the Synthesis of Demethylated Flavonols (Compounds 1–8)

To a stirring solution of 15 (0.500 g, 1.52 mmol) in anhydrous dichloromethane (30 mL) under nitrogen at 0 °C, boron tribromide in dichloromethane (1.0 M, 13.7 mL, 13.7 mmol, 9 equiv) was added. The mixture was allowed to warm at room temperature and was stirred for 2 days. The reaction mixture was then cooled to 0 °C, and methanol (10 mL) was added. The reaction mixture was concentrated in vacuo. Water (10 mL) was added, and the reaction sonicated and then left to stand. Compound 7 (0.440 g, quantitative yield) was collected as a red solid.

The procedure followed for the synthesis of all demethylated flavonols is analogue to that of compound 7 by considering 3 equiv of BBr₃ for each C–O bond cleavage.

Protein Expression and Purification

Recombinant TbPTR1 was expressed and purified by established methods (10) with minor revisions. The plasmid was supplied by Prof. William N. Hunter, University of Dundee. Briefly, protein expression was carried out in Escherichia coli BL21(DE3) cultured at 37 °C in SuperBroth (SB) medium to mid log phase and induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) overnight at 30 °C. (27) Cells were resuspended in 50 mM Tris-HCl, pH 7.5, 250 mM NaCl and disrupted by sonication. The target protein purification was achieved in a single step using a HisTrap FF 5 mL column (GE-Healthcare) and an imidazole concentration of 250 mM in the same buffer. The resulting protein sample was dialyzed overnight in 20 mM Tris-HCl pH 7.5 at 8 °C (membrane cutoff 10 kDa). The high purity of the sample was confirmed by SDS-PAGE analysis and MALDI-TOF mass spectrometry. The final protein yield of enzyme was established as approximately 50 mg/L bacterial culture.

The plasmid pET15b-LmPTR1 was supplied by Prof. William N. Hunter, University of Dundee. Protein expression was performed in the E. coli strain BL21(DE3) cultured at 37 °C in SB medium (supplemented with 100 mg/L ampicillin) to mid log phase and induced with 0.4 mM IPTG for 16 h at 28 °C. Cells, harvested by centrifugation, were resuspended in buffer A (50 mM Tris-HCl, pH 7.6, 20 mM imidazole, and 250 mM NaCl) and disrupted by sonication. The supernatant, collected by centrifugation, was further purified by nickel-affinity chromatography (HisTrap FF 5 mL column, GE-Healthcare), and the target protein was eluted using a 250–500 mM imidazole concentration in the same buffer. Fractions containing the protein (identified by SDS-PAGE) were pooled and combined with thrombin protease and then dialyzed overnight in 50 mM Tris-HCl, pH 7.6, at 25 °C. The mature protein was purified by a second nickel-affinity chromatography (HisTrap FF 5 mL column, GE-Healthcare) and eluted as weakly bound protein in 50–100 mM imidazole and 50 mM Tris-HCl, pH 7.6. The resulting protein sample was dialyzed in 20 mM sodium acetate pH 5.3 and 10 mM DTT. The high purity of the sample was confirmed by SDS-PAGE analysis and MALDI-TOF mass spectrometry, and the final protein yield established as approximately 30 mg/L bacterial culture.

Recombinant LmDHFR-TS was cloned in our lab. The gene coding sequence for LmDHFR-TS, cloned in pET-15b expression vector, was introduced by thermal shock into the E. coli strain ArcticExpress(DE3). Bacterial culture was grown at 30 °C in ZYP-5052 autoinduction medium (supplemented with ampicillin 100 mg/L) to OD_600 nm value of about 1, and then cell growth was continued for 60 h at 12 °C under vigorous aeration. Cells, harvested by centrifuge, were resuspended in buffer A (50 mM sodium citrate pH 5.5 and 0.25 M NaCl) and disrupted by sonication. The supernatant of the resulting crude extract was collected by centrifuge and purified by nickel-affinity chromatography. The target protein was eluted using a linear gradient 20–250 mM imidazole in the same buffer. Fractions containing the target protein (identified by SDS-PAGE) were pooled and dialyzed overnight in buffer A at 8 °C. The resulting protein sample was further purified on a gel filtration column (HiLoad 16/600 Superdex 200pg, GE Healthcare) equilibrated with 50 mM sodium citrate, pH 5.5, and 0.25 M NaCl). The high purity of the target protein was confirmed by SDS-PAGE analysis and the final yield established as approximately 10 mg/L bacterial culture.

The recombinant TbDHFR-TS was cloned in our lab. The synthetic gene encoding for TbDHFR-TS, optimized for E. coli expression and cloned in pET-15b expression vector, was purchased from GenScript USA Inc. The target protein was expressed and purified using the same protocol described for LmDHFR-TS, with minor modification. Briefly, cells were resuspended in buffer A (50 mM phosphate buffer, pH 7.5, 0.25 M NaCl, 10% glycerol, and 20 mM imidazole) and disrupted by sonication. The supernatant of the resulting crude extract was collected by centrifuge and purified by nickel-affinity chromatography (HisTrap FF 5 mL column, GE-Healthcare). The target protein was eluted using a 400 mM imidazole concentration in the same buffer. Eluted fractions were pooled and dialyzed overnight in buffer A at 8 °C. The resulting protein sample was concentrated and applied to a gel filtration column (HiLoad 16/600 Superdex 200pg, GE Healthcare) equilibrated in buffer A. The high purity of the sample was confirmed by SDS-PAGE and the final protein yield established as approximately 8 mg/L bacterial culture. hTS and hDHFR were purified as reported in literature. (15, 44)

The kinetic characterization (K_m and K_cat) of all the proteins purified and employed for the target/off target screening is reported in Supporting Information, Table S16.

X-ray Crystallography

Crystals of histidine-tagged TbPTR1 were obtained by the vapor diffusion hanging drop method technique at room temperature. (45) Drops were prepared by mixing equal volumes of protein (6–10 mg/mL in 20 mM Tris-HCl, pH 7.5, and 5–10 mM DTT) and precipitant (1.5–2.5 M sodium acetate and 0.1 M sodium citrate pH 5) solutions and equilibrated over a 600 μL reservoir. Well-ordered monoclinic crystals grew in 3–6 days. The TbPTR1–cofactor–inhibitor complexes were obtained by soaking crystals with each compound solubilized either in 1,4-dioxane or DMSO (without exceeding a dioxane-DMSO/crystal solution ratio of 1:9). After 4–21 h, crystals were transferred to a cryoprotectant, prepared by adding 30% v/v glycerol to the precipitant solution, and flash frozen in liquid nitrogen. Diffraction data for the complexes with compound NP-29 and NP-13 were measured using synchrotron radiation at the Diamond Light Source (DLS, Didcot, United Kingdom) beamline I04 equipped with a Pilatus 6M-F detector, using a wavelength of 0.9795 Å, a 0.25° rotation, and a 0.4 s exposure time/image. Data for the complex TbPTR1-NADPH/NADP⁺–2 were collected at the DLS beamline I03 equipped with a Pilatus3 6 M detector and using a wavelength of 0.9173 Å, a 0.25° rotation, and a 0.15 s exposure time/image. Data relative to the complex with compound 7 were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) beamline ID30A-1, equipped with a Dectris Pilatus3 2 M detector and by using a wavelength of 0.9660 Å, a 0.1° rotation, and a 0.08 s exposure time/image. X-ray images were integrated using Mosflm and scaled with SCALA within the CCP4 software suite. (46-48)TbPTR1 crystals belong to the monoclinic space group P2₁, showing only slight variations in cell parameters among the different crystals. Data collection and processing statistics are reported in Supporting Information, Table S2. The structures were solved by molecular replacement using the software Molrep (49) and a whole tetramer of TbPTR1 (PDB code 2X9G) as searching model. (50) Structural models were refined using REFMAC5, (51) whereas the program Coot (52) was used for electron density inspection, model manipulation, and placement of the solvent molecules. The final models were inspected manually and checked with the programs Coot and Procheck. (53) Data refinement statistics are reported in Supporting Information, Table S3. Figures were generated with CCP4 mg. (54)

TbPTR1, LmPTR1, TbDHFR, LmDHFR, hTS, and hDHFR Target Enzyme Assays

The in vitro assay used for PTR1 enzymes is based on the coupled assay reported by Shanks et al. (55) PTR1 uses H2B as a substrate and requires also NADPH for the reaction, and the reduction of H2B to H4B by PTR1 is nonenzymatically linked with the reduction of cytochrome c in this assay, which is detected at 550 nm. The formation of cyt c Fe²⁺ results in a signal increase in the photometric readout.

TbPTR1 and LmPTR1 activity was assayed in a buffer containing 20 mM sodium citrate (pH 6.0). The final reaction mixture contained the test compound at a range of concentrations and TbPTR1/LmPTR1 (6.0 nM/12 nM), H2B (0.3 μM/3 μM), cytochrome c (100 μM/100 μM), and NADPH (500 μM/500 μM). The final assay volume was 50 μL in 384-well clear plates (Greiner Bio-One, 781101). Compound screening was performed by addition of compound to assay plates (in 100% DMSO) followed by addition of 45 μL of Reaction Mix (enzyme, H2B cytochrome c in 20 mM sodium citrate buffer). A preread was made at 550 nm using an EnVision Multilabel Reader 2103 (PerkinElmer Inc., US), followed by incubation of the assay plates at 30 °C for 10 min. The reaction was initiated by the addition of 5 μL of NADPH (5 mM in ultrapure water), followed by the kinetically reading of the assay plates at 550 nm using the EnVision MultilabelReader at 10, 20, 30, 40, and 50 min, and the slope of each assay well was calculated. The screening data was analyzed using ActivityBase (IDBS) and for outlier elimination in the control wells the 3-σ method was applied. On the basis of the slope, data was normalized to the positive control methotrexate for TbPTR1/LmPTR1 (1 μM/50 μM, yielding 100% inhibition) and negative controls (1% DMSO, yielding 0% inhibition) and % inhibition calculated for all samples. The measurement at time 0 min was used to flag optically interfering samples. Each compound was tested in triplicate and the pIC₅₀ value, standard deviation, Hill slope, minimum signal, and maximum signal for each dose–response curve were obtained using a four-parameter logistic fit in the XE module of ActivityBase (IDBS).

LmDHFR, hDHFR, and hTS activities were assessed spectrophotometrically. (13) The K_m for the substrates and k_cat for all enzymes purified and used for the inhibition studies are reported in Supporting Information, Table S16.

In Vitro Biological Assays

A cloned line of L. infantum (MOM/MA671TMAP263) promastigotes were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 20 mM HEPES, and 1% Penn/Strep. Maintenance of promastigotes was done in T-25 flasks at 26 °C by subpassage at 10⁶ parasites/mL every 5–6 days. L. infantum axenic amastigotes expressing episomal luciferase were maintained in MAA/20 (axenic amastigote medium) at 37 °C under a 5% CO₂ environment with subpassages every 5 days. LUC-positive parasites were selected by addition of Geneticin sulfate (G418) to the culture at 60 μg/mL. Trypanosoma brucei brucei Lister 427 bloodstream forms were grown at 37 °C, 5% CO₂ in complete HMI-9 medium supplemented with 10% fetal calf serum (FCS) and 100 UI/mL of penicillin/streptomycin. Cultures were diluted before a cell density of 2 × 10⁶/mL was reached.

In Vitro Evaluation of Activity against L. infantum Intramacrophage Amastigotes

The efficacy of compounds against L. infantum intracellular amastigotes was determined according to literature (56) but with slight modifications. Briefly, 1 × 10⁶ THP-1-derived macrophages were infected with luciferase-expressing L. infantum axenic amastigotes in a macrophage:amastigotes ratio of 1:10 for 4 h at 37 °C, 5% CO₂. Noninternalized parasites were washed, and compounds were added at different concentrations (20–0.6 μM). After 72 h of incubation at 37 °C and 5% CO₂, media was substituted by PBS. Macrophages were lysed by addition of 25 μL of Glo-lysis buffer (Promega). Steady-Glo reagent (Promega) was then added, and the content of each well was transferred to white-bottom 96-well plates. Luminescence intensity was read using a Synergy 2 multi-mode reader (Biotek). The antileishmanial effect was evaluated by the determination of IC₅₀ value (concentration required to inhibit growth in 50%) and calculated by nonlinear regression analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, www.graphpad.com.

In Vitro Evaluation of Activity against T. brucei

The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified resazurin-based assay previously described. (57) Mid log bloodstream forms were added to an equal volume of serial dilutions of compounds in supplemented complete HMI-9 medium at a final cell density of 5 × 10³/mL. Following incubation for 72 h at 37 °C 5% CO₂, 20 μL of a 0.5 mM resazurin solution was added and plates were incubated for further 4 h under the same conditions. Fluorescence was measured at 540 and 620 nm excitation and emission wavelength, respectively, using a Synergy 2 multi-mode reader (Biotek). The antitrypanosomatid effect was evaluated by the determination of IC₅₀ value (concentration required to inhibit growth in 50%) and calculated by nonlinear regression analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California, www.graphpad.com. Half-maximal inhibitory concentrations (IC₅₀) reported correspond to the averages of the results obtained in at least two independent experiments. Compusyn software 1.0 was used to evaluate synergy. (34)

In Vitro Evaluation of Activity against T. cruzi

Infections were performed in 6-well plates (3 × 10⁶ HG39 cells/well). Confluent HG39 cells were infected with sanguineous trypomastigotes of T. cruzi Y strain at a 1:1 ratio. After 4 h, the cells were washed three times with PBS and fresh medium was added (RPMI supplemented with Glut/Pen/Strep and 20% heat-inactivated FCS, Sigma-Aldrich). The infected cells were incubated at 37 °C and 5% CO₂). Then 24 h post infection, the cells were treated with compounds and incubation was continued for 48 h. The gDNAs of infected cells were isolated using the LGC Genomics Mag Maxi kit following the manufacturer’s (LGC Genomics, Berlin) protocol and used as a template for a TaqMan probe-based quantitative real-time PCR. (58) The reference gene and the gene of interest were amplified using the KAPA Probe Fast qPCR Master Mix Kit (VWR) following the manufacturer protocol. Template DNA was used as 10% of the final reaction volume. Primers specific for human β-actin (reference gene) were human-β-AcF2 (CCCATCTACGAGGGGTATG) and human-β-AcR3 (TCGGTGAGGATCTTCATG). The parasite actin-specific primers were T. cruzi-AcF (CGTGAGAAGATGACACAG) and T. cruzi-AcR (GGGAGAGAGTATCCCTCG). All primers were used at a final concentration of 300 nM. The probe-specific for human β-actin was human-β-Ac probe (Cy5-CCTGGCTGGCCGGGACCTGAC-BHQ-3) labeled with the dye Cy5 at the 5′end and with the quencher BHQ-3 at the 3′ end. The probe specific for parasite actin was T. cruzi Ac probe (FAM-CACGCCATCACCAGCATCAAG-BHQ-1) labeled with the dye FAM at the 5′ end and with the quencher BHQ-1 at the 3′ end. Probes were used in a final concentration of 200 nM. The program was adapted to the use with the KAPA Probe Fast qPCR Master Mix Kit.

Cytotoxicity Assessment against THP-1 Macrophages

The effect of compounds 1–16 on THP-1-derived macrophages was assessed by the colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Briefly, 1 × 10⁶ THP-1 cells were differentiated into macrophages by addition of 20 ng/mL of phorbol-myristate 13-acetate (PMA, Sigma-Aldrich) for 18 h, followed by replacement with fresh medium for 24 h. Cells were incubated with compounds ranging from 100 to 3 μM after dilution in RPMI complete medium containing a maximum amount of 1% DMSO. After incubation for 72 h at 37 °C, 5% CO₂, medium was removed and a 0.5 mg/mL MTT solution was added. Plates were incubated for additional 4 h to allow viable cells to convert MTT into a purple formazan product. Solubilization of formazan crystals was achieved by addition of 2-propanol, and absorbance was read at 570 nm using a Synergy 2 multi-mode reader (Biotek). Cytotoxicity was evaluated by the determination of CC₅₀ value (drug concentration that reduced the percentage of viable cells in 50%) and calculated by nonlinear regression analysis using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California, www.graphpad.com.

hERG Cardiotoxicity Assay

The Invitrogen Predictor hERG fluorescence polarization assay was used in 384-well assay format (Greiner Bio-One, 784076) to test compounds. To each well of the assay plate, 100 nL of the test/control compound was added followed by addition of 5 μL of homogenized membrane solution (undiluted), followed by a further addition of 5 μL of tracer (1 nM final concentration in assay). The plates were incubated for 2 h at 25 °C in a humidity controlled incubator and the fluorescence polarization measured using an EnVision multilabel reader 2103 (Perkin Elmer Inc., US). The screening data was analyzed using ActivityBase (IDBS), and for outlier elimination in the control wells the 3-σ method was applied. The negative controls (0% inhibition) and positive controls E-4031, a blocker of hERG-type potassium channels (yielding 100% inhibition), were used to normalize the raw data. Each compound was tested in triplicate, and the pIC₅₀ value, standard deviation, Hill slope, minimum signal, and maximum signal for each dose–response curve were obtained using a four-parameter logistic fit in the XE module of ActivityBase (IDBS). All IC₅₀ data were associated with <10% SD.

Cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 Assay

The luminescence based P450-Glo (Promega) was used in 384-well assay format (Greiner Bio-One, 784076) to test compounds. The selected cytochrome P450 panel included microsomal preparations of cytochromes P450 1A2, 2C9, 2C19, 2D6, and 3A4 (Corning) from baculovirus infected insect cells (BTI-TN-5B1–4) which express cytochromes P450 (CYP) and cytochrome c reductase (and cytochrome b5 for 3A4). Compounds/controls were added into the empty 384-well plate (100 nL/well v/v 100% DMSO) using the Echo 550 Liquid Handler (Labcyte Inc., US), followed by addition of 5 μL/well of CYP/Luciferin substrate, and incubated for 30 min at 37 °C. The reaction was initiated by addition of 5 μL/well NADPH regeneration mixture. After 30 min at 37 °C, the CYP reaction was stopped and the luciferase reaction was initiated by addition of 10 μL/well of luciferin detection reagent. After additional 30 min at 37 °C, the luminescence readout was performed using the Infinite M1000 PRO plate reader (Tecan). Outliers in the control wells were eliminated according to the 3-σ method. Negative controls (0% inhibition) included only vehicle (v/v 1% DMSO), standard specific CYP inhibitors [CYP 1A2 inhibitor α-naphtho-flavone (Sigma-Aldrich, 15 nM), CYP 2C9 inhibitor sulfaphenazole (Sigma-Aldrich, 67 nM), CYP 2C19 inhibitor troglitazone (Sigma-Aldrich, 3.2 μM), CYP 2D6 inhibitor quinidine (Sigma-Aldrich, 2 nM), and CYP 3A4 inhibitor ketoconazole (Sigma-Aldrich, 54 nM)] were used as positive controls (100% inhibition). Each compound was tested in triplicate, and the pIC₅₀ value, standard deviation, Hill slope, minimum signal, and maximum signal for each dose–response curve were obtained using a four-parameter logistic fit in the XE module of ActivityBase (IDBS). All IC₅₀ data were associated with <10% SD.

Cytotoxicity Assay against A549 and WI38 Cells

These assays were performed using the CellTiter-Glo assay from Promega Corp. A549 cells obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and WI-38 cells were obtained from ATCC (ATCC CCL-75) and were grown on surface modified T175 cell culture flasks (cat. no. 660175, Greiner Bio-One GmbH, AT) in DMEM (cat. no. 41966–052, Life Technologies, US) with 10% FCS, streptomycin (100 μg/mL) and 100 U/mL penicillin G (cat. no. P-11-010, PAA Laboratories GmbH, AT). Cells were incubated at 37 °C in the presence of 5% CO₂ and were harvested at 80–90% confluency. Each test compound (200 nL of 10 mM in 100% v/v DMSO) was added to polystyrene 384-well cell culture microtiter plates (cat. no. 781073, 384 CellStar, Greiner Bio One, AT) using the Echo 550 Liquid Handler (Labcyte Inc., US). To harvest the cells, 1.5 mL of trypsin/EDTA (cat. no. L11-004, 0.5 and 0.22 mg/mL, respectively, PAA Laboratories GmbH, AT) was added per T175 flask and incubated at 37 °C in the presence of 5% CO₂ for 2 min. Detached cells were then resuspended in prewarmed media to a density of 0.2 × 10⁶ cells/mL. From this cell suspension, 20 μL was added per well of a 384-well microtiter plate, thereby giving a final test compound concentration of 100 μM and 0.1% v/v DMSO. After 48 h of incubation at 37 °C in the presence of 5% CO₂, 20 μL of CellTiter-Glo reagent (cat. no. G7571, Promega Inc., US) was added per well and the plate placed upon a linear shaker for 1 min at room temperature and further incubated at room temperature without shaking for 10 min. The luminescence readout was performed using the EnVision multilabel reader 2103 (PerkinElmer Inc., US) with a 0.5 s read time per well. Each assay plate also contained 16 wells for the positive control (cells prepared by treatment with cisplatin: 200 nL of 300 mM stock solution in 100% v/v DMSO, giving a final concentration of 3 mM cisplatin). The 16 wells for the negative controls per assay plate were prepared by treatment of cells with DMSO only (200 nL). Each compound was tested in triplicate, and the pIC₅₀ value, standard deviation, Hill slope, minimum signal, and maximum signal for each dose–response curve were obtained using a 4-parameter logistic fit in the XE module of ActivityBase (IDBS).

Assessment of Mitochondrial Toxicity

The 786-O (renal carcinoma) cell line was used for mitochondrial toxicity screening. Cells were maintained using RPMI-1640 medium containing 2 mM glutamine, 100 U/mL penicillin G, 100 mg/mL streptomycin, and 10% FCS. Cells were counted and diluted to a concentration of 75000 cells/mL, and 20 μL of this suspension (1500 cells/well) were added to Cell Carrier 384-well TC plates (PerkinElmer Inc.) and incubated for 36 h at 37 °C and 5% CO₂. Compound dilutions were prepared using a 384-well PP predilution plate (Greiner Bio-One), and 20 μL of the dilution were transferred to the 786-O cells. The low controls were valinomycin (final concentration of 1 μM and 1% DMSO v/v in column 23) and the high controls were 1% DMSO v/v in column 24. After 6 h incubation at 37 °C and 5% CO₂, 10 μL of a 200 nM solution of MitoTracker Red CMXRos (final concentration 50 nM) in prewarmed cell culture media (RPMI-1640 supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin) was added to each well and the 786-O cells were incubated for an additional 45 min at 37 °C and 5% CO₂. MitoTracker Red CMXRos uptake was measured using an Opera HCS system (PerkinElmer), and image analysis was performed using Columbus 2.4.0 (Perkin Elmer). All screening experiments were performed for the compounds as 11-point dose–responses in triplicates. The raw data were processed using GraphPad Prism (GraphPad Software Inc.), with outliers in control wells eliminated according to the 3-σ method. The dose–response curves were analyzed using four-parameter sigmoidal fit to obtain plausible results in the case of nonsigmoidal dose–response curves.

Aurora B Kinase Assay

The assay used was the ADP-Glo Kinase Enzyme System from PromegaCorp. The assay was performed in 384-well white low volume plate (Corning Inc., NY, 3824). The control compound used was SU6656 (Calbiochem, Germany, 572635) at a final concentration of 1 μM in column 23 and DMSO in column 24 at the same concentration (v/v) as the SU6656 control and the compound area. DMSO concentration is tolerated up to 2% final (v/v). An enzyme Mastermix containing 1× buffer, 50 μM DTT, and 17.5 ng/μL (35 μL/well) Aurora B [all reagents provided in the kit] was prepared. A substrate Mastermix containing 1× buffer, 36 μM ATP, and 7.5 ng/μL (15 ng/well) MBP protein as substrate [buffer, MBP provided in the Aurora B Kinase Enzyme System kit; ultrapure ATP provided in the ADP-Glo Kinase Assay kit] was prepared. Then 2 μL of the enzyme Mastermix and 2 μL of the substrate Mastermix were added to each well of a 384 low volume plate, and the plate was sealed using Thermowell sealing tape (Corning Inc. NY, USA) and incubated for 45 min at RT. The enzymatic reaction was stopped by adding 4 μL of ADP-Glo reagent [provided in the ADP-Glo Kinase Assay kit], and the plate is sealed again using Thermowell sealing tape (Corning Inc. NY) and incubated for 40 min at RT. Afterward, 8 μL of detection reagent were added to each well and the plate was sealed again. The plate was incubated for 45 min at RT, and afterward luminescence was measured using the EnVision multilabel reader 2103 (Perkin Elmer Inc., US). Each compound was tested in triplicate, and the pIC₅₀ value, standard deviation, Hill slope, minimum signal, and maximum signal for each dose–response curve were obtained using a four-parameter logistic fit in the XE module of ActivityBase (IDBS). All IC₅₀ data were associated with <10% SD.

Solubility Assays

UV–visible spectroscopy was used to measure the solubility of compound 2. The experiments were carried out with the instrument CARY 50. Six different solutions (3.125, 6.25, 12.5, 25, 50, and 100 μM) were prepared. The maximum of absorbance was found at 334 nm, and epsilon was calculated. Four saturated solutions were prepared: 100 μM in water, 100 μM in PBS, 200 μM in PBS + 10% DMSO, and 10 mM in PBS + 50% DMSO. The suspensions were left in incubation at 25 °C for 18 h, then filtered (0.2 μm). The absorbance at 334 nm was measured. One blank and three replicates of the same sample were performed for each solution. Using the calibration curve (Y = 0.0143x + 0.0033), we calculated the maximum solubility. Maximum solubility in water: 2.6 μM. Max solubility in PBS: 0.5 μM. Max solubility in PBS + 10% DMSO = 85.5 μM. Max solubility in PBS + 50% DMSO = 7.0 mM.

Compound 2 Encapsulation in PLGA and Solubilization with Cyclodextrins

The encapsulation of compound 2 on PLGA nanoparticles was obtained by a nanoprecipitation methodology. The polymer was dissolved in acetone at 20 mg/kg to for the diffusing phase. Then 0.5 mg of free compound were added for drug loading. This phase was then added to 10 mL of Pluronic F127 (0.1% in water) under moderate magnetic stirring at room temperature. For the evaporation of the organic solvent, the emulsion was stirred for 2 h. The formed NPs were recovered and washed by centrifugation and stored in PBS at 4 °C until use. Quantification of incorporated compound was done by the indirect method using absorption spectrophotometry. Standard curves for each compound were performed using 334 nm. The encapsulation efficiency was calculated as the ratio between the initial quantity of compound added to produce the NPs and the quantity of nonencapsulated compound present in the recovery and washing supernatants over the initial quantity of compound added to produce the NPs. Particles were characterized by dynamic light scattering (DLS); the size, polydispersity index, and ζ potential were determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) with a detection angle of 173°. Measurements were made in triplicate at 25 °C. Compound 2 was solubilized with hydroxypropyl-β-cyclodextrin (50% w/vol)/(Cavasol W7 HP Pharma, Ashland).

Pharmacokinetics of Compound 2

Plasma samples from 6 BALB/c mice treated with compound 2 administered alone (1 mg/kg IV) (5, 15, and 30 min post administration, pa), in PLGA nanoparticles (1 mg/kg/IV and oral) (0, 5, 15, 30, 45, 60 min and then 1 min, 3 min, 24 h, 48 h, and 72 h pa) and solubilized with cyclodextrins (NMRI mice, 4 mg/kg/per os) (1, 6, and 24 h pa) were analyzed by HPLC. Analysis was carried out with a modular Jasco HPLC fitted with a Hypersil BDS C18 column (250 mm × 4.6 mm, 5 μm) (Thermo Scientific) as stationary phase. Samples were treated with acetonitrile (ACN), vortexed, centrifuged (15 min, 6000 rpm), filtered through a 0.45 μm filter (Pall GHP Acrodisc GF), and assayed. Mobile phase was ACN:pure water (35:65), flow rate was 1.5 mL/min, injection loop was 100 μL, and detection at a wavelength of 335 nm. Sulfate and glucuronide conjugation of compound 2 was tested. (59)

Ethics Statement

All experiments performed at IBMC were carried out in accordance with the IBMC and INEB Animal Ethics Committees and the Portuguese National Authorities for Animal Health guidelines, according to the statements on the directive 2010/63/EU of the European Parliament and of the Council.

The experimental design and housing conditions at UCM were approved by the Committee of Animal Experimentation (Universidad Complutense de Madrid) and regional authorities (Community of Madrid) (ref. PROEX 169/15). Experiments were carried out at the animal house with official identification code ES280790001164 following the 3Rs principles. Animal handling and sampling were performed by trained and officially qualified personnel.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00698.

Compound characterization checklist (XLS)
Molecular formula strings (XLSX)
Compound 1–TbPTR1 (PDB)
Compound 2–TbPTR1 (PDB)
Compound 3–TbPTR1 (PDB)
Compound 4–TbPTR1 (PDB)
Compound 5–TbPTR1 (PDB)
Compound 6–TbPTR1 (PDB)
Compound 7–TbPTR1 (PDB)
Compound 8–TbPTR1 (PDB)
Compound 9–TbPTR1 (PDB)
Compound 10–TbPTR1 (PDB)
Compound 11–TbPTR1 (PDB)
Compound 12–TbPTR1 (PDB)
Compound 13–TbPTR1 (PDB)
Compound 14–TbPTR1 (PDB)
Compound 15–TbPTR1 (PDB)
Compound 16–TbPTR1 (PDB)
Compound 1–LmPTR1 (PDB)
Compound 2–LmPTR1 (PDB)
Compound 3–LmPTR1 (PDB)
Compound 4–LmPTR1 (PDB)
Compound 5–LmPTR1 (PDB)
Compound 6–LmPTR1 (PDB)
Compound 7–LmPTR1 (PDB)
Compound 8–LmPTR1 (PDB)
Compound 9–LmPTR1 (PDB)
Compound 10–LmPTR1 (PDB)
Compound 11–LmPTR1 (PDB)
Compound 12–LmPTR1 (PDB)
Compound 13–LmPTR1 (PDB)
Compound 14–LmPTR1 (PDB)
Compound 15–LmPTR1 (PDB)
Compound 16–LmPTR1 (PDB)
General structures of the flavonoids; code, chemical structure, purity, and IC₅₀ values of the 38 natural compounds screened; X-ray crystallographic data; inhibitory activity of the synthetic flavonols against TbPTR1 and LmPTR1; docking analysis; sequence alignment of TbPTR1 and LmPTR1; inhibitory activity of the synthetic flavonols against hDHFR, TbDHFR and LmDHFR; ADME-Tox data; antiparasitic activity of the synthesized compounds alone and in combination with MTX and synergy coefficients; isobologram and dose–effect curves for the combination of MTX and compound 13; toxicity of the combination on THP-1 cells; plasma concentration of compound 2 in BALB/c mice; nanoparticles characterization by dynamic light scattering; crystal structures used for conserved water analysis in TbPTR1 and LmPTR1; kinetic characterization of PTR1 and DHFR; characterization of the compounds 1–16 and of the intermediates 17–24 (PDF)

Accession Codes

PDB code 5JCJ was used for docking of compounds 1–16 (TbPTR1). PDB code 1E92 was used for docking of compounds 1–16 (LmPTR1). The Protein Data Bank accession codes of the X-ray crystallographic structures of TbPTR1 in complex with NP-13, NP-29, 2, and 7 are 5JDC, 5JCX, 5JDI, and 5JCJ, respectively. Authors will release the atomic coordinates and experimental data upon article publication.

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Author Information

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Corresponding Authors
- Stefania Ferrari - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; Email: [email protected]
- Stefano Mangani - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy; Email: [email protected]
- Maria Paola Costi - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; Email: [email protected]
Authors
- Chiara Borsari - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Rosaria Luciani - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Cecilia Pozzi - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
- Ina Poehner - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany
- Stefan Henrich - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany
- Matteo Trande - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Anabela Cordeiro-da-Silva - Instituto de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal
- Nuno Santarem - Instituto de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal
- Catarina Baptista - Instituto de Investigação e Inovação em Saúde, Universidade do Porto and Institute for Molecular and Cell Biology, 4150-180 Porto, Portugal
- Annalisa Tait - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Flavio Di Pisa - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
- Lucia Dello Iacono - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
- Giacomo Landi - Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
- Sheraz Gul - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Markus Wolf - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Maria Kuzikov - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Bernhard Ellinger - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Jeanette Reinshagen - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Gesa Witt - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Philip Gribbon - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Manfred Kohler - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Oliver Keminer - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Birte Behrens - Fraunhofer Institute for Molecular Biology and Applied Ecology-ScreeningPort, Schnackenburgallee 114 D-22525, Hamburg, Germany
- Luca Costantino - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Paloma Tejera Nevado - Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany
- Eugenia Bifeld - Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany
- Julia Eick - Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany
- Joachim Clos - Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg, Germany
- Juan Torrado - Complutense University of Madrid, 28040 Madrid, Spain
- María D. Jiménez-Antón - Complutense University of Madrid, 28040 Madrid, Spain; Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain
- María J. Corral - Complutense University of Madrid, 28040 Madrid, Spain; Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain
- José M^a Alunda - Complutense University of Madrid, 28040 Madrid, Spain; Instituto de Investigación Hospital 12 de Octubre, 28041 Madrid, Spain
- Federica Pellati - Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy
- Rebecca C. Wade - Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany; Center for Molecular Biology (ZMBH), DKFZ-ZMBH Alliance, Heidelberg University, 69120 Heidelberg, Germany; Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University,69120 Heidelberg, Germany
Notes
The authors declare no competing financial interest.

Acknowledgment

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This project has received funding from the European Union’s Seventh Framework Programme for research, technological development, and demonstration under grant agreement no. 603240 (NMTrypI, New Medicine for Trypanosomatidic Infections http://www.nmtrypi.eu/). We acknowledge the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and the Diamond Light Source (DLS, Didcot, United Kingdom) for providing synchrotron-radiation facilities, and we thank all of the staff for assistance in using the beamlines. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement no. 283570). I.P., S.H., and R.C.W. gratefully acknowledge the support of the Klaus Tschira Foundation. We thank Zaheer-ul-Haq Qasmi for his contributions to the initial computational analysis of the natural product library which were done with the support of the Alexander von Humboldt Foundation at HITS. The authors acknowledge the COST Action CM1307, http://www.cost.eu/COST_Actions/cmst/CM1307 for the contribution to the discussion of the research results.

Abbreviations Used

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WHO	World Health Organization
NTDs	neglected tropical diseases
HAT	human African trypanosomiasis
DNDi	Drugs for Neglected Diseases Initiative
DHFR	dihydrofolate reductase
TbPTR1	Trypanosoma brucei pteridine reductase 1
LmPTR1	Leishmania major pteridine reductase 1
MTX	methotrexate
L. infantum	Leishmania infantum
L. donovani	Leishmania donovani
T. brucei	Trypanosoma brucei
T. cruzi	Trypanosoma cruzi
CDCl₃	deuterated trichloromethane
EtOH	ethanol
MeOH	methanol
NaOH	sodium hydroxide
TMS	trimethylsilane
DLS	dynamic light scattering
NPs	nanoparticles
PLGA	poly(lactic-co-glycolicacid)
IV	intravenous
PBS	phosphate-buffered saline
ACN	acetonitrile
FBS	fetal bovine serum
EC₅₀	half-maximal effective concentration
CC₅₀	half-maximal cytotoxicity concentration
THP1	human monocytic cell line
A549	human lung adenocarcinoma epithelial cell line
WI-38	fetal lung fibroblasts cell lines
DRC	dose–response curve

References

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This article references 59 other publications.

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Human American trypanosomiasis, commonly called Chagas disease, is one of the most neglected illnesses in the world and remains one of the most prevalent chronic infectious diseases of Latin America with thousands of new cases every year. The only treatments available have been introduced five decades ago. They have serious, undesirable side effects and disputed benefits in the chronic stage of the disease - a characteristic and debilitating cardiomyopathy and/or megavisceras. Several labs. have therefore focused their efforts in finding better drugs. Although recent years have brought new clin. trials, these are few and lack diversity in terms of drug mechanism of action, thus resulting in a weak drug discovery pipeline. This fragility has been recently exposed by the failure of two candidates; posaconazole and E1224, to sterilely cure patients in phase 2 clin. trials. Such setbacks highlight the need for continuous, novel and high quality drug discovery and development efforts to discover better and safer treatments. In this article we will review past and current findings on drug discovery for Trypanosoma cruzi made by academic research groups, industry and other research organizations over the last half century. We also analyze the current research landscape that is now better placed than ever to deliver alternative treatments for Chagas disease in the near future.
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Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
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The treatment options of leishmaniasis are limited and far from satisfactory. For more than 60 years, treatment of leishmaniasis has centered around pentavalent antimonials (Sb(v)). Widespread misuse has led to the emergence of Sb(v) resistance in the hyperendemic areas of North Bihar. Other antileishmanials could also face the same fate, especially in the anthroponotic cycle. The HIV/ visceral leishmaniasis (VL) coinfected patients are another potential source for the emergence of drug resistance. At present no molecular markers of resistance are available and the only reliable method for monitoring resistance of isolates is the technically demanding in vitro amastigote-macrophage model. As the armametrium of drugs for leishmaniasis is limited, it is important that effective monitoring of drug use and response should be done to prevent the spread of resistance. Regimens of simultaneous or sequential combinations should be seriously considered to limit the emergence of resistance.
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New models of drug discovery have been developed to overcome the lack of modern and effective drugs for neglected diseases such as human African trypanosomiasis (HAT; sleeping sickness), leishmaniasis, and Chagas disease, which have no financial viability for the pharmaceutical industry. With the purpose of combining the skills and research capacity in academia, pharmaceutical industry, and contract researchers, public-private partnerships or product development partnerships aim to create focused research consortia that address all aspects of drug discovery and development. These consortia not only emulate the projects within pharmaceutical and biotechnology industries, eg, identification and screening of libraries, medicinal chemistry, pharmacology and pharmacodynamics, formulation development, and manufacturing, but also use and strengthen existing capacity in disease-endemic countries, particularly for the conduct of clinical trials. The Drugs for Neglected Diseases initiative (DNDi) has adopted a model closely related to that of a virtual biotechnology company for the identification and optimization of drug leads. The application of this model to the development of drug candidates for the kinetoplastid infections of HAT, Chagas disease, and leishmaniasis has already led to the identification of new candidates issued from DNDi's own discovery pipeline. This demonstrates that the model DNDi has been implementing is working but its DNDi, neglected diseases sustainability remains to be proven.
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A review. As in centuries past, the main weapon against human malaria infections continues to be intervention with drugs, despite the widespread and increasing frequency of parasite populations that are resistant to one or more of the available compds. This is a particular problem with the lethal species of parasite, Plasmodium falciparum, which claims some two million lives per yr as well as causing enormous social and economic problems. Among the antimalarial drugs currently in clin. use, the antifolates have the best defined mol. targets, namely the enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), which function in the folate metabolic pathway. The products of this pathway, reduced folate cofactors, are essential for DNA synthesis and the metab. of certain amino acids. Moreover, their formation and interconversions involve a no. of other enzymes that have not as yet been exploited as drug targets. Antifolates are of major importance as they currently represent the only inexpensive regime for combating chloroquine-resistant malaria, and are now first-line drugs in a no. of African countries. Aspects of our understanding of this pathway and antifolate drug resistance are reviewed here, with a particular emphasis on approaches to analyzing the details of, and balance between, folate biosynthesis by the parasite and salvage of pre-formed folate from exogenous sources.
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Gilbert, Ian H.
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A review. The protozoan diseases leishmaniasis, Chagas' disease and African trypanosomiasis are major health problems in many countries, particularly developing countries, and there are few drugs available to treat these diseases. Dihydrofolate reductase (DHFR) inhibitors have been used successfully in the treatment of a no. of other diseases such as cancer, malaria and bacterial infections; however they have not been used for the treatment of these diseases. This article summarizes studies on leishmanial and trypanosomal DHFR inhibitor development and evaluation. Possible mechanisms of resistance to DHFR inhibitors are also discussed.
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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 (6) 1457– 1468 DOI: 10.1111/j.1365-2958.2006.05332.x
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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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Guerrieri, D.; Ferrari, S.; Costi, M. P.; Michels, P. A. Biochemical effects of riluzole on Leishmania parasites Exp. Parasitol. 2013, 133 (3) 250– 254 DOI: 10.1016/j.exppara.2012.11.013
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Biochemical effects of riluzole on Leishmania parasites
Guerrieri, Davide; Ferrari, Stefania; Costi, M. Paola; Michels, Paul A. M.
Experimental Parasitology (2013), 133 (3), 250-254CODEN: EXPAAA; ISSN:0014-4894. (Elsevier Inc.)
We have previously shown that riluzole (6-(trifluoromethoxy)benzothiazol-2-amine), an agent used to treat CNS disorders, possesses inhibitory activity against pteridine reductase (PTR1) in pathogenic protists at low micromolar concns. Therefore, the potential use of this drug in anti-parasitic chemotherapy deserves evaluation. In this study, we report the effect of this compd. on cell cultures of Leishmania mexicana and L. major. The anti-parasitic activity of riluzole was confirmed, with the largest effect obsd. when the drug was administered to cells during their exponential growth phase. Moreover, a remarkable decrease in PTR1 activity was obsd. in the lysates of cells pretreated with the compd., which is due to impairment of the enzyme's preferential reaction with biopterin as a cofactor. In addn., the treatment increased the parasites' susceptibility to oxidative stress, affecting the ability of Leishmania to survive under severe oxidative conditions. These results suggest that the inhibitory effect of riluzole on PTR1 is not the only mechanism through which it induces the death of Leishmania parasites.
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Barrack, K. L.; Tulloch, L. B.; Burke, L. A.; Fyfe, P. K.; Hunter, W. N. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2011, 67, 33– 37 DOI: 10.1107/S174430911004724X
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Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site
Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.
Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2011), 67 (1), 33-37CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein prodn. was prepd. and the enzyme was purified and crystd. in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resoln. and the structure was solved by mol. replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a crit. component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.
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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.; Wade, R. C.; Costi, M. P. Virtual screening identification of non folate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase J. Med. Chem. 2011, 54 (1) 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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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 (1) 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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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 (5) 1448– 1453 DOI: 10.1073/pnas.0704384105
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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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Balunas, M. J.; Kinghorn, A. D. Drug discovery from medicinal plants Life Sci. 2005, 78 (5) 431– 441 DOI: 10.1016/j.lfs.2005.09.012
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Drug discovery from medicinal plants
Balunas, Marcy J.; Kinghorn, A. Douglas
Life Sciences (2005), 78 (5), 431-441CODEN: LIFSAK; ISSN:0024-3205. (Elsevier B.V.)
A review. Current research in drug discovery from medicinal plants involves a multifaceted approach combining botanical, phytochem., biol., and mol. techniques. Medicinal plant drug discovery continues to provide new and important leads against various pharmacol. targets including cancer, HIV/AIDS, Alzheimer's, malaria, and pain. Several natural product drugs of plant origin have either recently been introduced to the United States market, including arteether, galanthamine, nitisinone, and tiotropium, or are currently involved in late-phase clin. trials. As part of our National Cooperative Drug Discovery Group (NCDDG) research project, numerous compds. from tropical rainforest plant species with potential anticancer activity have been identified. Our group has also isolated several compds., mainly from edible plant species or plants used as dietary supplements, that may act as chemopreventive agents. Although drug discovery from medicinal plants continues to provide an important source of new drug leads, numerous challenges are encountered including the procurement of plant materials, the selection and implementation of appropriate high-throughput screening bioassays, and the scale-up of active compds.
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Annang, F.; Pérez-Moreno, G.; García-Hernández, R.; Cordon-Obras, C.; Martín, J.; Tormo, J. R.; Rodríguez, L.; de Pedro, N.; Gómez-Pérez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Pérez, L. M.; Navarro, M.; Gamarro, F.; González-Pacanowska, D. High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease J. Biomol. Screening 2015, 20 (1) 82– 91 DOI: 10.1177/1087057114555846
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High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human african trypanosomiasis, leishmaniasis, and chagas disease
Annang, F.; Perez-Moreno, G.; Garcia-Hernandez, R.; Cordon-Obras, C.; Martin, J.; Tormo, J. R.; Rodriguez, L.; de Pedro, N.; Gomez-Perez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Perez, L. M.; Navarro, M.; Gamarro, F.; Pacanowska, D. Gonzalez
Journal of Biomolecular Screening (2015), 20 (1), 82-91, 10 pp.CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compds. against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chem. libraries using phenotypic and target-based approaches. Combinatorial chem. libraries contain hundreds of thousands of compds.; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chem. diversity that can serve as excellent templates for the synthesis of novel, biol. active mols. We report here a validated HTS platform for the screening of microbial exts. against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial exts. from the MEDINA Natural Products library. Tandem liq. chromatog.-mass spectrometry showed that 48 exts. contain potentially new compds. that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product exts. against the above-mentioned kinetoplastid parasites.
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Harvey, A. L. Natural products in drug discovery Drug Discovery Today 2008, 13 (19–20) 894– 901 DOI: 10.1016/j.drudis.2008.07.004
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Natural products in drug discovery
Harvey, Alan L.
Drug Discovery Today (2008), 13 (19/20), 894-901CODEN: DDTOFS; ISSN:1359-6446. (Elsevier B.V.)
A review. Natural products have been the single most productive source of leads for the development of drugs. Over a 100 new products are in clin. development, particularly as anti-cancer agents and anti-infectives. Application of mol. biol. techniques is increasing the availability of novel compds. that can be conveniently produced in bacteria or yeasts, and combinatorial chem. approaches are being based on natural product scaffolds to create screening libraries that closely resemble drug-like compds. Various screening approaches are being developed to improve the ease with which natural products can be used in drug discovery campaigns, and data mining and virtual screening techniques are also being applied to databases of natural products. It is hoped that the more efficient and effective application of natural products will improve the drug discovery process.
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Harvey, A. L.; Edrada-Ebel, R. A.; Quinn, R. J. The re-emergence of natural products for drug discovery in the genomics era Nat. Rev. Drug Discovery 2015, 14, 111– 129 DOI: 10.1038/nrd4510
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The re-emergence of natural products for drug discovery in the genomics era
Harvey, Alan L.; Edrada-Ebel, RuAngelie; Quinn, Ronald J.
Nature Reviews Drug Discovery (2015), 14 (2), 111-129CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
Natural products have been a rich source of compds. for drug discovery. However, their use has diminished in the past two decades, in part because of tech. barriers to screening natural products in high-throughput assays against mol. targets. Here, we review strategies for natural product screening that harness the recent tech. advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.
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Ndjonka, D.; Rapado, L. N.; Silber, A. M.; Liebau, E.; Wrenger, C. Natural Products as a Source for Treating Neglected Parasitic Diseases Int. J. Mol. Sci. 2013, 14, 3395– 3439 DOI: 10.3390/ijms14023395
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Natural products as a source for treating neglected parasitic diseases
Ndjonka, Dieudonne; Rapado, Ludmila Nakamura; Silber, Ariel M.; Liebau, Eva; Wrenger, Carsten
International Journal of Molecular Sciences (2013), 14 (), 3395-3439CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
A review. Infectious diseases caused by parasites are a major threat for the entire mankind, esp. in the tropics. More than 1 billion people world-wide are directly exposed to tropical parasites such as the causative agents of trypanosomiasis, leishmaniasis, schistosomiasis, lymphatic filariasis and onchocerciasis, which represent a major health problem, particularly in impecunious areas. Unlike most antibiotics, there is no "general" antiparasitic drug available. Here, the selection of antiparasitic drugs varies between different organisms. Some of the currently available drugs are chem. de novo synthesized, however, the majority of drugs are derived from natural sources such as plants which have subsequently been chem. modified to warrant higher potency against these human pathogens. In this review article we will provide an overview of the current status of plant derived pharmaceuticals and their chem. modifications to target parasite-specific peculiarities in order to interfere with their proliferation in the human host.
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Singh, N.; Mishra, B. B.; Bajpai, S.; Singh, R. K.; Tiwari, V. K. Natural product based leads to fight against leishmaniasis Bioorg. Med. Chem. 2014, 22 (1) 18– 45 DOI: 10.1016/j.bmc.2013.11.048
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Natural product based leads to fight against leishmaniasis
Singh, Nisha; Mishra, Bhuwan B.; Bajpai, Surabhi; Singh, Rakesh K.; Tiwari, Vinod K.
Bioorganic & Medicinal Chemistry (2014), 22 (1), 18-45CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
A review. The growing incidence of parasitic resistance against generic pentavalent antimonials, specifically for visceral disease in Indian subcontinent, is a serious issue in Leishmania control. Notwithstanding the two treatment alternatives, that is amphotericin B and miltefosine are being effectively used but their high cost and therapeutic complications limit their use in endemic areas. In the absence of a vaccine candidate, identification, and characterization of novel drugs and targets is a major requirement of leishmanial research. This review describes current drug regimens, putative drug targets, numerous natural products that have shown promising antileishmanial activity along with some key issues and strategies for future research to control leishmaniasis worldwide.
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Tasdemir, D.; Kaiser, M.; Brun, R.; Yardley, V.; Schmidt, T. J.; Tosun, F.; Rüedi, P. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies Antimicrob. Agents Chemother. 2006, 50 (4) 1352– 1364 DOI: 10.1128/AAC.50.4.1352-1364.2006
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Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies
Tasdemir, Deniz; Kaiser, Marcel; Brun, Reto; Yardley, Vanessa; Schmidt, Thomas J.; Tosun, Fatma; Ruedi, Peter
Antimicrobial Agents and Chemotherapy (2006), 50 (4), 1352-1364CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycons and glycosides, as well as a panel of other related compds. of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. The cytotoxicities of more than 100 compds. for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compds. were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concn. [IC50], 68 ng/mL), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3',4'-tetrahydroxyflavone (IC50s, 0.5 μg/mL) and catechol (IC50, 0.8 μg/mL). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC50s less than 5.0 μg/mL. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC50s of 0.6, 0.7, 0.8, and 1.0 μg/mL, resp. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compds. lacked cytotoxicity in vitro and in vivo. By screening a large no. of flavonoids and analogs, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quant. structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivs. as potent and effective antiprotozoal agents.
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da Silva, E. R.; do Carmo Maquiaveli, C.; Magalhães, P. P. The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase Exp. Parasitol. 2012, 130 (3) 183– 188 DOI: 10.1016/j.exppara.2012.01.015
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The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase
da Silva, Edson Roberto; Maquiaveli, Claudia do Carmo; Magalhaes, Prislaine Pupolin
Experimental Parasitology (2012), 130 (3), 183-188CODEN: EXPAAA; ISSN:0014-4894. (Elsevier Inc.)
Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compds., and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC50 values for quercetin, quercitrin and isoquercitrin were estd. to be 3.8, 10 and 4.3 μM, resp. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate L-arginine and the cofactor Mn2+ at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking anal. of these flavonols suggests that the cathecol group of the three compds. interact with Asp129, which is involved in metal bridge formation for the cofactors MnA2+ and MnB2+ in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.
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Manjolin, L. C.; dos Reis, M. B. G.; do Carmo Maquiaveli, C.; Santos-Filho, O. A.; da Silva, E. R. Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection Food Chem. 2013, 141 (3) 2253– 2262 DOI: 10.1016/j.foodchem.2013.05.025
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Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection
Manjolin, Leticia Correa; dos Reis, Matheus Balduino Goncalves; Maquiaveli, Claudia do Carmo; Santos-Filho, Osvaldo Andrade; da Silva, Edson Roberto
Food Chemistry (2013), 141 (3), 2253-2262CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
Fisetin, quercetin, luteolin, and 7,8-dihydroxyflavone show high arginase inhibitory activity in Leishmania cultures and have low toxicity for mammalian cells. The structural aspects of 13 flavonoids were analyzed for their inhibition of the arginase enzyme from Leishmania amazonensis. Higher arginase inhibition was obsd. with fisetin, which was 4- and 10-times greater than the inhibition by quercetin and luteolin, resp. The hydroxyl group at position 3 may contribute to the inhibitory activity toward arginase, while the hydroxyl group at position 5 may not. The absence of the catechol group on apigenin drastically decreased the arginase inhibition. The docking of compds. showed that the inhibitors interacted with amino acids involved in the Mn2+-Mn2+ metal bridge formation at the catalytic site. Due to the low IC50 values of these flavonoids, they may be used as food supplements in leishmaniasis treatment.
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Mamani-Matsuda, M.; Rambert, J.; Malvy, D.; Lejoly-Boisseau, H.; Daulouède, S.; Thiolat, D.; Coves, S.; Courtois, P.; Vincendeau, P.; Mossalayi, M. D. Quercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophages Antimicrob. Agents Chemother. 2004, 48 (3) 924– 929 DOI: 10.1128/AAC.48.3.924-929.2004
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Quercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophages
Mamani-Matsuda, Maria; Rambert, Jerome; Malvy, Denis; Lejoly-Boisseau, Helene; Daulouede, Sylvie; Thiolat, Denis; Coves, Sara; Courtois, Pierrette; Vincendeau, Philippe; Mossalayi, M. Djavad
Antimicrobial Agents and Chemotherapy (2004), 48 (3), 924-929CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
In addn. to parasite spread, the severity of disease obsd. in cases of human African trypanosomiasis (HAT), or sleeping sickness, is assocd. with increased levels of inflammatory mediators, including tumor necrosis factor (TNF)-α and nitric oxide derivs. In the present study, quercetin (3,3',4',5,7-pentahydroxyflavone), a potent immunomodulating flavonoid, was shown to directly induce the death of Trypanosoma brucei gambiense, the causative agent of HAT, without affecting normal human cell viability. Quercetin directly promoted T. b. gambiense death by apoptosis as shown by Annexin V binding. In addn. to microbicidal activity, quercetin induced dose-dependent decreases in the levels of TNF-α and nitric oxide produced by activated human macrophages. These results highlight the potential use of quercetin as an antimicrobial and anti-inflammatory agent for the treatment of African trypanosomiasis.
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Dias, T. A.; Duarte, C. L.; Lima, C. F.; Proença, M. F.; Pereira-Wilson, C. Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin Eur. J. Med. Chem. 2013, 65, 500– 510 DOI: 10.1016/j.ejmech.2013.04.064
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Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin
Dias, Tatiana A.; Duarte, Cecilia L.; Lima, Cristovao F.; Proenca, M. Fernanda; Pereira-Wilson, Cristina
European Journal of Medicinal Chemistry (2013), 65 (), 500-510CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
A series of chalcones I [R1 = H, HO, MeO; R2 = H, Me, MeO, Br; R3 = H, Me, MeO, etc.; R4, R5 = H, MeO] and flavonols II [R1 = H, MeO; R2 = H, Me, MeO, Br; R3 = H, Me, MeO, etc.; R4 = H, MeO] were synthesized in good yields by an eco-friendly approach. A pharmacol. evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed that the anticancer activity of flavonols was higher when compared with that of the resp. chalcone precursors. The antiproliferative activity of halogenated derivs. increases as the substituent in the 3- or 4-position of the B-ring goes from F to Cl and to Br. In addn., halogens in position 3 enhance anticancer activity in chalcones whereas for flavonol derivs. the best performance was registered for the 4-substituted derivs. Flow cytometry anal. showed that 2 compds. induced cell cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were corroborated by western blot and fluorescence microscopy anal. In summary, halogenated chalcones and flavonols were successfully prepd. and presented high anticancer activity as shown by their cell growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a pos. control.
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Juvale, K.; Stefan, K.; Wiese, M. Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2 Eur. J. Med. Chem. 2013, 67, 115– 126 DOI: 10.1016/j.ejmech.2013.06.035
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Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2
Juvale, Kapil; Stefan, Katja; Wiese, Michael
European Journal of Medicinal Chemistry (2013), 67 (), 115-126CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting mols. with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a soln. to overcome MDR. The authors synthesized a series of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 3' and 4' of the (benzo)flavone structure. All synthesized compds. were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compds. were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-assocd. protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addn. most active compds. were investigated for their cytotoxicity. It was obsd. that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH3 substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3',4'-OCH3 on Ph ring lead to increase in activity as compared to other substituents. Compd. 24, a 7,8-benzoflavone deriv. was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concns.
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Setzer, W. N.; Ogungbe, I. V. In-silico Investigation of Antitrypanosomal Phytochemicals from Nigerian Medicinal Plants PLoS Neglected Trop. Dis. 2012, 6 (7) e1727 DOI: 10.1371/journal.pntd.0001727
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In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plants
Setzer, William N.; Ogungbe, Ifedayo V.
PLoS Neglected Tropical Diseases (2012), 6 (7), e1727CODEN: PNTDAM; ISSN:1935-2735. (Public Library of Science)
Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment. A mol. docking study has been carried out on phytochem. agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacol. to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compds. from 19 species of medicinal plants were investigated using in-silico mol. docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4' epimerase (TbUDPGE), and ornithine decarboxylase (TbODC). This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4' epimerase (TbUDPGE). Polyphenolic compds. such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins. This in-silico mol. docking study has identified potential biomol. targets of phytochem. components of antitrypanosomal plants and has detd. which phytochem. classes and structural manifolds likely target trypanosomal enzymes. The results could provide the framework for synthetic modification of bioactive phytochems., de novo synthesis of structural motifs, and lead to further phytochem. investigations.
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Kavanagh, K. L.; Jornvall, H.; Persson, B.; Oppermann, U. Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes Cell. Mol. Life Sci. 2008, 65, 3895– 3906 DOI: 10.1007/s00018-008-8588-y
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The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes
Kavanagh, K. L.; Joernvall, H.; Persson, B.; Oppermann, U.
Cellular and Molecular Life Sciences (2008), 65 (24), 3895-3906CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
A review. Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have crit. roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metab. as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an α/β folding pattern with a central beta sheet flanked by 2-3 α-helixes from each side, thus a classical Rossmann fold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is detd. by a variable C-terminal segment. Relationships exist with bacterial haloalc. dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signaling.
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Gourley, D. G.; Schuttelkopf, 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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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 (6) 1457– 1468 DOI: 10.1111/j.1365-2958.2006.05332.x
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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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Chatelain, E. Chagas disease drug discovery: toward a new era J. Biomol. Screening 2015, 20 (1) 22– 35 DOI: 10.1177/1087057114550585
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Chagas disease drug discovery: toward a new era
Chatelain, Eric
Journal of Biomolecular Screening (2015), 20 (1), 22-35, 14 pp.CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
A review. American trypanosomiasis, or Chagas disease, is the result of infection by the Trypanosoma cruzi parasite. Endemic in Latin America where it is the major cause of death from cardiomyopathy, the impact of the disease is reaching global proportions through migrating populations. New drugs that are safe, efficacious, low cost, and adapted to the field are critically needed. Over the past five years, there has been increased interest in the disease and a surge in activities within various organizations. However, recent clin. trials with azoles, specifically posaconazole and the ravuconazole prodrug E1224, were disappointing, with treatment failure in Chagas patients reaching 70% to 90%, as opposed to 6% to 30% failure for benznidazole-treated patients. The lack of translation from in vitro and in vivo models to the clinic obsd. for the azoles raises several questions. There is a scientific requirement to review and challenge whether we are indeed using the right tools and decision-making processes to progress compds. forward for the treatment of this disease. New developments in the Chagas field, including new technologies and tools now available, will be discussed, and a redesign of the current screening strategy during the discovery process is proposed.
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Katsuno, K.; Burrows, J. N.; Duncan, K.; Hooft van Huijsduijnen, R.; Kaneko, T.; Kita, K.; Mowbray, C. E.; Schmatz, D.; Warner, P.; Slingsby, B. T. Hit and lead criteria in drug discovery for infectious diseases of the developing world Nat. Rev. Drug Discovery 2015, 14 (11) 751– 758 DOI: 10.1038/nrd4683
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Hit and lead criteria in drug discovery for infectious diseases of the developing world
Katsuno, Kei; Burrows, Jeremy N.; Duncan, Ken; van Huijsduijnen, Rob Hooft; Kaneko, Takushi; Kita, Kiyoshi; Mowbray, Charles E.; Schmatz, Dennis; Warner, Peter; Slingsby, B. T.
Nature Reviews Drug Discovery (2015), 14 (11), 751-758CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
Reducing the burden of infectious diseases that affect people in the developing world requires sustained collaborative drug discovery efforts. The quality of the chem. starting points for such projects is a key factor in improving the likelihood of clin. success, and so it is important to set clear go/no-go criteria for the progression of hit and lead compds. With this in mind, the Japanese Global Health Innovative Technol. (GHIT) Fund convened with experts from the Medicines for Malaria Venture, the Drugs for Neglected Diseases initiative and the TB Alliance, together with representatives from the Bill & Melinda Gates Foundation, to set disease-specific criteria for hits and leads for malaria, tuberculosis, visceral leishmaniasis and Chagas disease. Here, we present the agreed criteria and discuss the underlying rationale.
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Chou, T. C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies Pharmacol. Rev. 2006, 58 (3) 621– 681 DOI: 10.1124/pr.58.3.10
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Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies
Chou, Ting-Chao
Pharmacological Reviews (2006), 58 (3), 621-681CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
A review. The median-effect equation derived from the mass-action law principle at equil. steady state via math. induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theor. basis for the combination index (CI)-isobologram equation that allows quant. detn. of drug interactions, where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, resp. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-redn. index plot, and polygonogram for in vitro or in vivo studies. This theor. development, exptl. design, and computerized data anal. have facilitated dose-effect anal. for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of exptl. designs and real data anal. are also illustrated. The merging of the mass-action law principle with math. induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compd. or entity to how to beneficially use multiple drugs or modalities in combination therapies.
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Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation J. Mol. Biol. 1995, 245, 43– 53 DOI: 10.1016/S0022-2836(95)80037-9
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Molecular recognition of a receptor sites using a genetic algorithm with a description of desolvation
Jones, Gareth; Willett, Peter; Glen, Robert C.
Journal of Molecular Biology (1995), 245 (1), 43-53CODEN: JMOBAK; ISSN:0022-2836. (Academic)
Understanding the principles whereby macromol. biol. receptors can recognize small mol. substrates or inhibitors is the subject of a major effort. This is of paramount importance in rational drug design where the receptor structure is known (the "docking" problem). Current theor. approaches utilize models of the steric and electrostatic interaction of bound ligands and recently conformational flexibility has been incorporated. The authors report results based on software using a genetic algorithm that uses an evolutionary strategy in exploring the full conformational flexibility of the ligand with partial flexibility of the protein, and which satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Results are reported on five test systems showing excellent agreement with exptl. data. The design of the algorithm offers insight into the mol. recognition mechanism.
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Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking J. Mol. Biol. 1997, 267, 727– 748 DOI: 10.1006/jmbi.1996.0897
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Development and validation of a genetic algorithm for flexible docking
Jones, Gareth; Willett, Peter; Glen, Robert C.; Leach, Andrew R.; Taylor, Robin
Journal of Molecular Biology (1997), 267 (3), 727-748CODEN: JMOBAK; ISSN:0022-2836. (Academic)
Prediction of small mol. binding modes to macromols. of known three-dimensional structure is a problem of paramount importance in rational drug design (the "docking" problem). We report the development and validation of the program GOLD (Genetic Optimization for Ligand Docking). GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein and satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Numerous enhancements and modifications have been applied to the original technique resulting in a substantial increase in the reliability and the applicability of the algorithm. The advanced algorithm has been tested on a dataset of 100 complexes extd. from the Brookhaven Protein Data Bank. When used to dock the ligand back into the binding site, GOLD achieved a 71% success rate in identifying the exptl. binding mode.
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Schrödinger Release 2015-4: Maestro, version 10.4; Schrödinger, LLC: New York, 2015.
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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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Small-Molecule Drug Discovery Suite 2015-4: Glide, version 6.9; Schrödinger, LLC: New York, 2015.
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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 Enclosure 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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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.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shaw, D. E.; Shelley, M.; Perry, J. K.; 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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Li, H.; Robertson, A. D.; Jensen, J. H. Very Fast Empirical Prediction and Interpretation of Protein pKa Values Proteins: Struct., Funct., Genet. 2005, 61, 704– 721 DOI: 10.1002/prot.20660
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Very fast empirical prediction and rationalization of protein pKa values
Li, Hui; Robertson, Andrew D.; Jensen, Jan H.
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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Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, O. M. H.; 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 (7) 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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Leslie, A. G. The integration of macromolecular diffraction data Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48– 57 DOI: 10.1107/S0907444905039107
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The integration of macromolecular diffraction data
Leslie, Andrew G. W.
Acta Crystallographica, Section D: Biological Crystallography (2006), D62 (1), 48-57CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
The objective of any modern data-processing program is to produce from a set of diffraction images a set of indexes (hkls) with their assocd. intensities (and ests. of their uncertainties), together with an accurate est. of the crystal unit-cell parameters. This procedure should not only be reliable, but should involve an abs. min. of user intervention. The process can be conveniently divided into three stages. The first (autoindexing) dets. the unit-cell parameters and the orientation of the crystal. The unit-cell parameters may indicate the likely Laue group of the crystal. The second step is to refine the initial est. of the unit-cell parameters and also the crystal mosaicity using a procedure known as post-refinement. The third step is to integrate the images, which consists of predicting the positions of the Bragg reflections on each image and obtaining an est. of the intensity of each reflection and its uncertainty. This is carried out while simultaneously refining various detector and crystal parameters. Basic features of the algorithms employed for each of these three sep. steps are described, principally with ref. to the program MOSFLM.
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Evans, P. Scaling and assessment of data quality Acta Crystallogr., Sect. 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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Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760– 763. DOI: 10.1107/S0907444994003112
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Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement J. Appl. Crystallogr. 1997, 30, 1022– 1025 DOI: 10.1107/S0021889897006766
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MOLREP: an automated program for molecular replacement
Vagin, Alexei; Teplyakov, Alexei
Journal of Applied Crystallography (1997), 30 (6), 1022-1025CODEN: JACGAR; ISSN:0021-8898. (Munksgaard International Publishers Ltd.)
MOLREP is an automated program for mol. replacement which uses effective new approaches in data processing and rotational and translational searching. These include an automatic choice of all parameters, scaling by Patterson origin peaks and soft resoln. cutoff. One of the cornerstones of the program is an original full-symmetry translation function combined with a packing function. Information from the model already placed in the cell is incorporated in both translation and packing functions. A no. of tests using exptl. data proved the ability of the program to find the correct soln. in difficult cases.
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Dawson, A.; Tulloch, L. B.; Barrack, K. L.; Hunter, W. N. High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 1334– 1340 DOI: 10.1107/S0907444910040886
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High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target
Dawson, Alice; Tulloch, Lindsay B.; Barrack, Keri L.; Hunter, William N.
Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (12), 1334-1340CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resoln. structures have been detd., using data between 1.6 and 1.1 Å resoln., of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine deriv. The structures provide insight into the interactions formed by new mol. entities in the enzyme active site with ligands that represent lead compds. for structure-based inhibitor development and to support early-stage drug discovery.
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Murshudov, G. N.; Vagin, A.; Dodson, E. J. Refinement of macromolecular structures by themaximum-likelihood method Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240– 255 DOI: 10.1107/S0907444996012255
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Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics Acta Crystallogr., Sect. 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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Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M.PROCHECK - a program to check the stereochemical quality of protein structures J. Appl. Crystallogr. 1993, 26, 283– 291 DOI: 10.1107/S0021889892009944
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PROCHECK: a program to check the stereochemical quality of protein structures
Laskowski, Roman A.; MacArthur, Malcolm W.; Moss, David S.; Thornton, Janet M.
Journal of Applied Crystallography (1993), 26 (2), 283-91CODEN: JACGAR; ISSN:0021-8898.
The PROCHECK suite of programs provides a detailed check on the stereochem. of a protein structure. Its outputs comprise a no. of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well-refined structures of the same resoln. and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modeled on known structures.
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McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E. M. Presenting your structures: the CCP4mg molecular-graphics software Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 386– 394 DOI: 10.1107/S0907444911007281
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Presenting your structures: The CCP4mg molecular-graphics software
McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E. M.
Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 386-394CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
CCP4mg is a mol.-graphics program that is designed to give rapid access to both straightforward and complex static and dynamic representations of macromol. structures. It has recently been updated with a new interface that provides more sophisticated atom-selection options and a wizard to facilitate the generation of complex scenes. These scenes may contain a mixt. of coordinate-derived and abstr. graphical objects, including text objects, arbitrary vectors, geometric objects and imported images, which can enhance a picture and eliminate the need for subsequent editing. Scene descriptions can be saved to file and transferred to other mols. Here, the substantially enhanced version 2 of the program, with a new underlying GUI toolkit, is described. A built-in rendering module produces publication-quality images.
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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 (2) 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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Sereno, D.; Cavaleyra, M.; Zemzoumi, K.; Maquaire, S.; Ouaissi, A.; Lemesre, J. L. Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action Antimicrob. Agents Chemother. 1998, 42 (12) 3097– 3102
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Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action
Sereno D; Cavaleyra M; Zemzoumi K; Maquaire S; Ouaissi A; Lemesre J L
Antimicrobial agents and chemotherapy (1998), 42 (12), 3097-102 ISSN:0066-4804.
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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Bowling, T.; Mercer, L.; Don, R.; Jacobs, R.; Nare, B. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis Int. J. Parasitol.: Drugs Drug Resist. 2012, 2, 262– 270 DOI: 10.1016/j.ijpddr.2012.02.002
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Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis
Bowling Tana; Mercer Luke; Jacobs Robert; Nare Bakela; Don Robert
International journal for parasitology. Drugs and drug resistance (2012), 2 (), 262-70 ISSN:2211-3207.
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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Bifeld, E.; Tejera Nevado, P.; Bartsch, J.; Eick, J.; Clos, J. A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues. Med. Microbiol. Immunol. 2016, epub ahead of print.
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Shia, C.; Tsai, S.; Kuo, S.; Hou, Y.; Chao, P. L. Metabolism and pharmacokinetics of 3,3‘,4‘,7- tetrahydeoxyflavone (fisetin), 5-hydroxyflavone and 7-hydroxyflavone and antihemolysis effects of fisetin and its serum metabolites J. Agric. Food Chem. 2009, 57, 83– 89 DOI: 10.1021/jf802378q
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Metabolism and Pharmacokinetics of 3,3',4',7-Tetrahydroxyflavone (Fisetin), 5-Hydroxyflavone, and 7-Hydroxyflavone and Antihemolysis Effects of Fisetin and Its Serum Metabolites
Shia, Chi-Sheng; Tsai, Shang-Yuan; Kuo, Sheng-Chu; Hou, Yu-Chi; Chao, Pei-Dawn Lee
Journal of Agricultural and Food Chemistry (2009), 57 (1), 83-89CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
3,3',4',7-Tetrahydroxyflavone (fisetin) has shown various beneficial bioactivities. This study investigated the metab. and pharmacokinetics of fisetin, 5-hydroxyflavone (5-OH-flavone), and 7-hydroxyflavone (7-OH-flavone) in male Sprague-Dawley rats. Blood was withdrawn via cardiopuncture and assayed by HPLC before and after hydrolysis with sulfatase and β-glucuronidase. The results indicated that after i.v. administration of fisetin (10 mg/kg of bw), fisetin declined rapidly and fisetin sulfates/glucuronides emerged instantaneously. When fisetin (50 mg/kg of bw) was given orally, fisetin parent form was transiently present in serum only during the absorption phase, whereas fisetin sulfates/glucuronides predominated. The serum metabolites of fisetin showed less potent inhibition on 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced hemolysis than fisetin. Following oral administrations of 40 mg/kg of bw of 5-OH-flavone and 7-OH-flavone, the glucuronide of 5-OH-flavone and the sulfate/glucuronide of 7-OH-flavone were found in serum, whereas no traces of parent forms were detected. In conclusion, fisetin and 7-OH-flavone were rapidly and extensively biotransformed into their sulfate/glucuronide, whereas 5-OH-flavone was exclusively metabolized to glucuronide.
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Abstract
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Figure 1
Figure 1. Activity profile of the 38 phytochemicals screened against TbPTR1, hTS, and hDHFR and against the T. brucei parasite. The IC₅₀ is indicated by the color: dark-green, 0–30 μM; green, 31–90 μM; light-green, 90–150 μM; yellow, 151–250 μM; red, >250 μM; gray, not tested. *, Catechins; **, triterpenes; ***, anthraquinones.
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Figure 2
Figure 2. Crystal structures of TbPTR1 (gray cartoon, interacting residues in sticks) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms) and four inhibitors (in sticks) (A) NP-29 (cyan), (B) NP-13 (orange), (C) compound 2 (lilac), and (D) compound 7 (yellow). Hydrogen bond interactions (dashed lines) in the active site are shown. The 2F_o – F_c electron density maps corresponding to the inhibitors (dark-blue wire) and NADPH/NADP⁺ (light-blue wire), contoured at the 1σ level are shown. The chemical structures and atom names of the ligands are specified in the insets.
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Figure 3
Figure 3. Design of the synthetic library (left). NP-13 (in yellow) into TbPTR1 (in gray). The amino acids involved in the interactions of the designed compounds are shown in different colors. For clarity reasons, two residues involved in the interactions are not shown (Cys168 and Asn175). Superimposition of the four crystal structures of TbPTR1 (right) (chain A gray ribbon; chain D pink ribbon; relevant active site residues as light-blue sticks) in complex with NADPH/NADP⁺ (sticks, black carbon atoms) and the inhibitors (stick) NP-13 (orange), NP-29 (cyan), compound 2 (lilac), and compound 7 (yellow). The three different binding modes adopted by the ligands can be appreciated as well as the movement of Trp221 (yellow sticks) upon binding of compound 7.
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Scheme 1
Scheme 1. Synthesis of the Flavonols 1–16^a
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Scheme aReaction conditions: (a) NaOH (3 M), EtOH, rt; (b) H₂O₂, NaOH (1 M), EtOH, rt; (c) BBr₃ (1 M in dry DMC), dry DMC, 0 °C → rt.
Figure 4
Figure 4. Inhibitory activity against TbPTR1 (in gray) and LmPTR1 (in black). The control compound was pyrimethamine, a PTR1 inhibitor (100% inhibition at 50 μM against both PTR1 enzymes).
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Figure 5
Figure 5. Comparison of the effects of hydroxyl substituents at the R6 (left) and the R7 (right) positions of ring A on the binding of the synthetic flavonoids to TbPTR1. Superimposition of constraint docking poses for compound 2 (in sticks, lilac carbons) and compound 5 (in sticks, pale-green carbons) (left) and compound 3 (in sticks, orange carbons) and compound 6 (in sticks, brown carbons) (right) in TbPTR1 (in cartoon with interacting residues in sticks with gray carbons) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.
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Figure 6
Figure 6. Comparison of the effects of hydroxyl and methoxy substituents at the R6 (left) and the R3′ (left and right) positions on the binding of the synthetic flavonoids to TbPTR1. Constraint docking poses are shown for compound 2 (in sticks, lilac carbons), compound 10 (in sticks, dark-green carbons) (left), compound 9 (in sticks, pale-cyan carbons), and compound 1 (in sticks, magenta carbons) (right) in TbPTR1 (in cartoon with interacting residues in sticks with gray carbons) in complex with NADPH/NADP⁺ (in sticks, black carbon atoms). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.
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Figure 7
Figure 7. Superimposition of the crystal structure of LmPTR1 (PDB ID 1E92) in cartoon representation and interacting residues in sticks representation. Chains A and D (containing Arg287) are colored in pale-pink and magenta, respectively) and the best predicted receptor conformation obtained in the induced-fit docking study starting from this crystal structure (His241 in H-bonding contact to compound 7) in complex with NADPH/NADP⁺ (in sticks, black carbons) and compound 7 (in sticks, yellow carbons). A conserved water molecule is shown in ball-and-stick representation. Hydrogen bonds are indicated by dark-gray dotted lines.
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Figure 8
Figure 8. Early toxicity properties combined with inhibitory activity against T. brucei. The data are reported as a traffic light system. An ideal compound (I. comp.) should have all the parameters green. The cells are colored in green when the percentage of inhibition of T. brucei and the percentage of A549 and W1–38 cell growth is between 60 and 100, while the percentage of inhibition of CYP isoforms, hERG, Aurora B kinase and mitochondrial toxicity is between 0 and 30. Cells are colored in red when data indicates toxicity or inactivity. Yellow stands for a borderline value (30–60%): moderately active or slightly toxic compound. Gray: not tested.
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Figure 9
Figure 9. Antiparasitic activity of the synthesized compounds against Trypanosoma brucei (in gray), Trypanosoma cruzi (in green), and Leishmania infantum (in black) at 10 μM. The reference compounds were pentamidine (IC₅₀ = 1.55 ± 0.24 nM) for T. brucei, miltefosine (IC₅₀ = 2.65 ± 0.4 μM) for L. infantum, and nifurtimox (IC₅₀ = 2.2 ± 0.4 μM) for T. cruzi.
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Figure 10
Figure 10. Trypanocidal activity of eight synthesized compounds alone and in combination with MTX. The compounds were tested at three different concentrations: 5 μM (in green), 2.5 μM (in light-pink), 1.25 μM (in light-blue). Antiparasitic activity of MTX is shown in dark-red.
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  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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3. 3
  Gaspar, L.; Moraes, C. B.; Freitas-Junior, L. H.; Ferrari, S.; Costantino, L.; Costi, M. P.; Coron, R. P.; Smith, T.; Siqueira-Neto, J. L.; McKerrow, J.; Cordeiro-da-Silva, A. Current and Future Chemotherapy For Chagas Disease Curr. Med. Chem. 2015, 22 (37) 4293– 4312 DOI: 10.2174/0929867322666151015120804
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  3
  Current and Future Chemotherapy for Chagas Disease
  Gaspar, Luis; Moraes, Carolina B.; Freitas-Junior, Lucio H.; Ferrari, Stefania; Costantino, Luca; Costi, Maria Paola; Coron, Ross P.; Smith, Terry K.; Siqueira-Neto, Jair L.; McKerrow, James H.; Cordeiro-da-Silva, Anabela
  Current Medicinal Chemistry (2015), 22 (37), 4293-4312CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
  Human American trypanosomiasis, commonly called Chagas disease, is one of the most neglected illnesses in the world and remains one of the most prevalent chronic infectious diseases of Latin America with thousands of new cases every year. The only treatments available have been introduced five decades ago. They have serious, undesirable side effects and disputed benefits in the chronic stage of the disease - a characteristic and debilitating cardiomyopathy and/or megavisceras. Several labs. have therefore focused their efforts in finding better drugs. Although recent years have brought new clin. trials, these are few and lack diversity in terms of drug mechanism of action, thus resulting in a weak drug discovery pipeline. This fragility has been recently exposed by the failure of two candidates; posaconazole and E1224, to sterilely cure patients in phase 2 clin. trials. Such setbacks highlight the need for continuous, novel and high quality drug discovery and development efforts to discover better and safer treatments. In this article we will review past and current findings on drug discovery for Trypanosoma cruzi made by academic research groups, industry and other research organizations over the last half century. We also analyze the current research landscape that is now better placed than ever to deliver alternative treatments for Chagas disease in the near future.
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4. 4
  Barrett, M. P.; Vincent, I. M.; Burchmore, R. J.; Kazibwe, A. J.; Matovu, E. Drug resistance in human African trypanosomiasis Future Microbiol. 2011, 6, 1037– 1047 DOI: 10.2217/fmb.11.88
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  4
  Drug resistance in human African trypanosomiasis
  Barrett Michael P; Vincent Isabel M; Burchmore Richard J S; Kazibwe Anne J N; Matovu Enock
  Future microbiology (2011), 6 (9), 1037-47 ISSN:.
  Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
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5. 5
  Chakravarty, J.; Sundar, S. Drug resistance in Leishmaniasis J. Glob. Infect. Dis. 2010, 2, 167– 176 DOI: 10.4103/0974-777X.62887
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  5
  Drug resistance in leishmaniasis
  Chakravarty Jaya; Sundar Shyam
  Journal of global infectious diseases (2010), 2 (2), 167-76 ISSN:.
  The treatment options of leishmaniasis are limited and far from satisfactory. For more than 60 years, treatment of leishmaniasis has centered around pentavalent antimonials (Sb(v)). Widespread misuse has led to the emergence of Sb(v) resistance in the hyperendemic areas of North Bihar. Other antileishmanials could also face the same fate, especially in the anthroponotic cycle. The HIV/ visceral leishmaniasis (VL) coinfected patients are another potential source for the emergence of drug resistance. At present no molecular markers of resistance are available and the only reliable method for monitoring resistance of isolates is the technically demanding in vitro amastigote-macrophage model. As the armametrium of drugs for leishmaniasis is limited, it is important that effective monitoring of drug use and response should be done to prevent the spread of resistance. Regimens of simultaneous or sequential combinations should be seriously considered to limit the emergence of resistance.
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6. 6
  Chatelain, E.; Ioset, J.-R. Drug discovery and development for neglected diseases: the DNDi model Drug Des., Dev. Ther. 2011, 5, 175– 181 DOI: 10.2147/DDDT.S16381
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  6
  Drug discovery and development for neglected diseases: the DNDi model
  Chatelain Eric; Ioset Jean-Robert
  Drug design, development and therapy (2011), 5 (), 175-81 ISSN:.
  New models of drug discovery have been developed to overcome the lack of modern and effective drugs for neglected diseases such as human African trypanosomiasis (HAT; sleeping sickness), leishmaniasis, and Chagas disease, which have no financial viability for the pharmaceutical industry. With the purpose of combining the skills and research capacity in academia, pharmaceutical industry, and contract researchers, public-private partnerships or product development partnerships aim to create focused research consortia that address all aspects of drug discovery and development. These consortia not only emulate the projects within pharmaceutical and biotechnology industries, eg, identification and screening of libraries, medicinal chemistry, pharmacology and pharmacodynamics, formulation development, and manufacturing, but also use and strengthen existing capacity in disease-endemic countries, particularly for the conduct of clinical trials. The Drugs for Neglected Diseases initiative (DNDi) has adopted a model closely related to that of a virtual biotechnology company for the identification and optimization of drug leads. The application of this model to the development of drug candidates for the kinetoplastid infections of HAT, Chagas disease, and leishmaniasis has already led to the identification of new candidates issued from DNDi's own discovery pipeline. This demonstrates that the model DNDi has been implementing is working but its DNDi, neglected diseases sustainability remains to be proven.
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7. 7
  Gilbert, I. H. Drug discovery for neglected diseases: molecular target-based and phenotypic approaches J. Med. Chem. 2013, 56 (20) 7719– 7726 DOI: 10.1021/jm400362b
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  7
  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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8. 8
  Hyde, J. E. Exploring the folate pathway in Plasmodium falciparum Acta Trop. 2005, 94 (3) 191– 206 DOI: 10.1016/j.actatropica.2005.04.002
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  8
  Exploring the folate pathway in Plasmodium falciparum
  Hyde, John E.
  Acta Tropica (2005), 94 (3), 191-206CODEN: ACTRAQ; ISSN:0001-706X. (Elsevier B.V.)
  A review. As in centuries past, the main weapon against human malaria infections continues to be intervention with drugs, despite the widespread and increasing frequency of parasite populations that are resistant to one or more of the available compds. This is a particular problem with the lethal species of parasite, Plasmodium falciparum, which claims some two million lives per yr as well as causing enormous social and economic problems. Among the antimalarial drugs currently in clin. use, the antifolates have the best defined mol. targets, namely the enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), which function in the folate metabolic pathway. The products of this pathway, reduced folate cofactors, are essential for DNA synthesis and the metab. of certain amino acids. Moreover, their formation and interconversions involve a no. of other enzymes that have not as yet been exploited as drug targets. Antifolates are of major importance as they currently represent the only inexpensive regime for combating chloroquine-resistant malaria, and are now first-line drugs in a no. of African countries. Aspects of our understanding of this pathway and antifolate drug resistance are reviewed here, with a particular emphasis on approaches to analyzing the details of, and balance between, folate biosynthesis by the parasite and salvage of pre-formed folate from exogenous sources.
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9. 9
  Gilbert, I. H. Inhibitors of dihydrofolate reductase in Leishmania and trypanosomes Biochim. Biophys. Acta, Mol. Basis Dis. 2002, 1587 (2–3) 249– 257 DOI: 10.1016/S0925-4439(02)00088-1
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  9
  Inhibitors of dihydrofolate reductase in leishmania and trypanosomes
  Gilbert, Ian H.
  Biochimica et Biophysica Acta, Molecular Basis of Disease (2002), 1587 (2-3), 249-257CODEN: BBADEX; ISSN:0925-4439. (Elsevier B.V.)
  A review. The protozoan diseases leishmaniasis, Chagas' disease and African trypanosomiasis are major health problems in many countries, particularly developing countries, and there are few drugs available to treat these diseases. Dihydrofolate reductase (DHFR) inhibitors have been used successfully in the treatment of a no. of other diseases such as cancer, malaria and bacterial infections; however they have not been used for the treatment of these diseases. This article summarizes studies on leishmanial and trypanosomal DHFR inhibitor development and evaluation. Possible mechanisms of resistance to DHFR inhibitors are also discussed.
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10. 10
  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 (6) 1457– 1468 DOI: 10.1111/j.1365-2958.2006.05332.x
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  10
  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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11. 11
  Guerrieri, D.; Ferrari, S.; Costi, M. P.; Michels, P. A. Biochemical effects of riluzole on Leishmania parasites Exp. Parasitol. 2013, 133 (3) 250– 254 DOI: 10.1016/j.exppara.2012.11.013
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  11
  Biochemical effects of riluzole on Leishmania parasites
  Guerrieri, Davide; Ferrari, Stefania; Costi, M. Paola; Michels, Paul A. M.
  Experimental Parasitology (2013), 133 (3), 250-254CODEN: EXPAAA; ISSN:0014-4894. (Elsevier Inc.)
  We have previously shown that riluzole (6-(trifluoromethoxy)benzothiazol-2-amine), an agent used to treat CNS disorders, possesses inhibitory activity against pteridine reductase (PTR1) in pathogenic protists at low micromolar concns. Therefore, the potential use of this drug in anti-parasitic chemotherapy deserves evaluation. In this study, we report the effect of this compd. on cell cultures of Leishmania mexicana and L. major. The anti-parasitic activity of riluzole was confirmed, with the largest effect obsd. when the drug was administered to cells during their exponential growth phase. Moreover, a remarkable decrease in PTR1 activity was obsd. in the lysates of cells pretreated with the compd., which is due to impairment of the enzyme's preferential reaction with biopterin as a cofactor. In addn., the treatment increased the parasites' susceptibility to oxidative stress, affecting the ability of Leishmania to survive under severe oxidative conditions. These results suggest that the inhibitory effect of riluzole on PTR1 is not the only mechanism through which it induces the death of Leishmania parasites.
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12. 12
  Barrack, K. L.; Tulloch, L. B.; Burke, L. A.; Fyfe, P. K.; Hunter, W. N. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2011, 67, 33– 37 DOI: 10.1107/S174430911004724X
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  12
  Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site
  Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.
  Acta Crystallographica, Section F: Structural Biology and Crystallization Communications (2011), 67 (1), 33-37CODEN: ACSFCL; ISSN:1744-3091. (International Union of Crystallography)
  Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein prodn. was prepd. and the enzyme was purified and crystd. in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resoln. and the structure was solved by mol. replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a crit. component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes.
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13. 13
  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.; Wade, R. C.; Costi, M. P. Virtual screening identification of non folate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase J. Med. Chem. 2011, 54 (1) 211– 221 DOI: 10.1021/jm1010572
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  13
  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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14. 14
  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 (1) 221– 229 DOI: 10.1021/jm901059x
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  14
  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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15. 15
  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 (5) 1448– 1453 DOI: 10.1073/pnas.0704384105
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  15
  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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16. 16
  Balunas, M. J.; Kinghorn, A. D. Drug discovery from medicinal plants Life Sci. 2005, 78 (5) 431– 441 DOI: 10.1016/j.lfs.2005.09.012
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  16
  Drug discovery from medicinal plants
  Balunas, Marcy J.; Kinghorn, A. Douglas
  Life Sciences (2005), 78 (5), 431-441CODEN: LIFSAK; ISSN:0024-3205. (Elsevier B.V.)
  A review. Current research in drug discovery from medicinal plants involves a multifaceted approach combining botanical, phytochem., biol., and mol. techniques. Medicinal plant drug discovery continues to provide new and important leads against various pharmacol. targets including cancer, HIV/AIDS, Alzheimer's, malaria, and pain. Several natural product drugs of plant origin have either recently been introduced to the United States market, including arteether, galanthamine, nitisinone, and tiotropium, or are currently involved in late-phase clin. trials. As part of our National Cooperative Drug Discovery Group (NCDDG) research project, numerous compds. from tropical rainforest plant species with potential anticancer activity have been identified. Our group has also isolated several compds., mainly from edible plant species or plants used as dietary supplements, that may act as chemopreventive agents. Although drug discovery from medicinal plants continues to provide an important source of new drug leads, numerous challenges are encountered including the procurement of plant materials, the selection and implementation of appropriate high-throughput screening bioassays, and the scale-up of active compds.
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17. 17
  Annang, F.; Pérez-Moreno, G.; García-Hernández, R.; Cordon-Obras, C.; Martín, J.; Tormo, J. R.; Rodríguez, L.; de Pedro, N.; Gómez-Pérez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Pérez, L. M.; Navarro, M.; Gamarro, F.; González-Pacanowska, D. High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease J. Biomol. Screening 2015, 20 (1) 82– 91 DOI: 10.1177/1087057114555846
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  17
  High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human african trypanosomiasis, leishmaniasis, and chagas disease
  Annang, F.; Perez-Moreno, G.; Garcia-Hernandez, R.; Cordon-Obras, C.; Martin, J.; Tormo, J. R.; Rodriguez, L.; de Pedro, N.; Gomez-Perez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Perez, L. M.; Navarro, M.; Gamarro, F.; Pacanowska, D. Gonzalez
  Journal of Biomolecular Screening (2015), 20 (1), 82-91, 10 pp.CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
  African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compds. against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chem. libraries using phenotypic and target-based approaches. Combinatorial chem. libraries contain hundreds of thousands of compds.; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chem. diversity that can serve as excellent templates for the synthesis of novel, biol. active mols. We report here a validated HTS platform for the screening of microbial exts. against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial exts. from the MEDINA Natural Products library. Tandem liq. chromatog.-mass spectrometry showed that 48 exts. contain potentially new compds. that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product exts. against the above-mentioned kinetoplastid parasites.
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18. 18
  Harvey, A. L. Natural products in drug discovery Drug Discovery Today 2008, 13 (19–20) 894– 901 DOI: 10.1016/j.drudis.2008.07.004
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  18
  Natural products in drug discovery
  Harvey, Alan L.
  Drug Discovery Today (2008), 13 (19/20), 894-901CODEN: DDTOFS; ISSN:1359-6446. (Elsevier B.V.)
  A review. Natural products have been the single most productive source of leads for the development of drugs. Over a 100 new products are in clin. development, particularly as anti-cancer agents and anti-infectives. Application of mol. biol. techniques is increasing the availability of novel compds. that can be conveniently produced in bacteria or yeasts, and combinatorial chem. approaches are being based on natural product scaffolds to create screening libraries that closely resemble drug-like compds. Various screening approaches are being developed to improve the ease with which natural products can be used in drug discovery campaigns, and data mining and virtual screening techniques are also being applied to databases of natural products. It is hoped that the more efficient and effective application of natural products will improve the drug discovery process.
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19. 19
  Harvey, A. L.; Edrada-Ebel, R. A.; Quinn, R. J. The re-emergence of natural products for drug discovery in the genomics era Nat. Rev. Drug Discovery 2015, 14, 111– 129 DOI: 10.1038/nrd4510
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  19
  The re-emergence of natural products for drug discovery in the genomics era
  Harvey, Alan L.; Edrada-Ebel, RuAngelie; Quinn, Ronald J.
  Nature Reviews Drug Discovery (2015), 14 (2), 111-129CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
  Natural products have been a rich source of compds. for drug discovery. However, their use has diminished in the past two decades, in part because of tech. barriers to screening natural products in high-throughput assays against mol. targets. Here, we review strategies for natural product screening that harness the recent tech. advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.
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  Ndjonka, D.; Rapado, L. N.; Silber, A. M.; Liebau, E.; Wrenger, C. Natural Products as a Source for Treating Neglected Parasitic Diseases Int. J. Mol. Sci. 2013, 14, 3395– 3439 DOI: 10.3390/ijms14023395
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  20
  Natural products as a source for treating neglected parasitic diseases
  Ndjonka, Dieudonne; Rapado, Ludmila Nakamura; Silber, Ariel M.; Liebau, Eva; Wrenger, Carsten
  International Journal of Molecular Sciences (2013), 14 (), 3395-3439CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
  A review. Infectious diseases caused by parasites are a major threat for the entire mankind, esp. in the tropics. More than 1 billion people world-wide are directly exposed to tropical parasites such as the causative agents of trypanosomiasis, leishmaniasis, schistosomiasis, lymphatic filariasis and onchocerciasis, which represent a major health problem, particularly in impecunious areas. Unlike most antibiotics, there is no "general" antiparasitic drug available. Here, the selection of antiparasitic drugs varies between different organisms. Some of the currently available drugs are chem. de novo synthesized, however, the majority of drugs are derived from natural sources such as plants which have subsequently been chem. modified to warrant higher potency against these human pathogens. In this review article we will provide an overview of the current status of plant derived pharmaceuticals and their chem. modifications to target parasite-specific peculiarities in order to interfere with their proliferation in the human host.
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  Singh, N.; Mishra, B. B.; Bajpai, S.; Singh, R. K.; Tiwari, V. K. Natural product based leads to fight against leishmaniasis Bioorg. Med. Chem. 2014, 22 (1) 18– 45 DOI: 10.1016/j.bmc.2013.11.048
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  21
  Natural product based leads to fight against leishmaniasis
  Singh, Nisha; Mishra, Bhuwan B.; Bajpai, Surabhi; Singh, Rakesh K.; Tiwari, Vinod K.
  Bioorganic & Medicinal Chemistry (2014), 22 (1), 18-45CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
  A review. The growing incidence of parasitic resistance against generic pentavalent antimonials, specifically for visceral disease in Indian subcontinent, is a serious issue in Leishmania control. Notwithstanding the two treatment alternatives, that is amphotericin B and miltefosine are being effectively used but their high cost and therapeutic complications limit their use in endemic areas. In the absence of a vaccine candidate, identification, and characterization of novel drugs and targets is a major requirement of leishmanial research. This review describes current drug regimens, putative drug targets, numerous natural products that have shown promising antileishmanial activity along with some key issues and strategies for future research to control leishmaniasis worldwide.
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22. 22
  Tasdemir, D.; Kaiser, M.; Brun, R.; Yardley, V.; Schmidt, T. J.; Tosun, F.; Rüedi, P. Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies Antimicrob. Agents Chemother. 2006, 50 (4) 1352– 1364 DOI: 10.1128/AAC.50.4.1352-1364.2006
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  22
  Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies
  Tasdemir, Deniz; Kaiser, Marcel; Brun, Reto; Yardley, Vanessa; Schmidt, Thomas J.; Tosun, Fatma; Ruedi, Peter
  Antimicrobial Agents and Chemotherapy (2006), 50 (4), 1352-1364CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
  Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycons and glycosides, as well as a panel of other related compds. of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani. The cytotoxicities of more than 100 compds. for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compds. were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concn. [IC50], 68 ng/mL), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3',4'-tetrahydroxyflavone (IC50s, 0.5 μg/mL) and catechol (IC50, 0.8 μg/mL). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC50s less than 5.0 μg/mL. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC50s of 0.6, 0.7, 0.8, and 1.0 μg/mL, resp. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compds. lacked cytotoxicity in vitro and in vivo. By screening a large no. of flavonoids and analogs, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quant. structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivs. as potent and effective antiprotozoal agents.
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23. 23
  da Silva, E. R.; do Carmo Maquiaveli, C.; Magalhães, P. P. The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase Exp. Parasitol. 2012, 130 (3) 183– 188 DOI: 10.1016/j.exppara.2012.01.015
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  23
  The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase
  da Silva, Edson Roberto; Maquiaveli, Claudia do Carmo; Magalhaes, Prislaine Pupolin
  Experimental Parasitology (2012), 130 (3), 183-188CODEN: EXPAAA; ISSN:0014-4894. (Elsevier Inc.)
  Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compds., and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC50 values for quercetin, quercitrin and isoquercitrin were estd. to be 3.8, 10 and 4.3 μM, resp. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate L-arginine and the cofactor Mn2+ at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking anal. of these flavonols suggests that the cathecol group of the three compds. interact with Asp129, which is involved in metal bridge formation for the cofactors MnA2+ and MnB2+ in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.
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24. 24
  Manjolin, L. C.; dos Reis, M. B. G.; do Carmo Maquiaveli, C.; Santos-Filho, O. A.; da Silva, E. R. Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection Food Chem. 2013, 141 (3) 2253– 2262 DOI: 10.1016/j.foodchem.2013.05.025
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  24
  Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection
  Manjolin, Leticia Correa; dos Reis, Matheus Balduino Goncalves; Maquiaveli, Claudia do Carmo; Santos-Filho, Osvaldo Andrade; da Silva, Edson Roberto
  Food Chemistry (2013), 141 (3), 2253-2262CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)
  Fisetin, quercetin, luteolin, and 7,8-dihydroxyflavone show high arginase inhibitory activity in Leishmania cultures and have low toxicity for mammalian cells. The structural aspects of 13 flavonoids were analyzed for their inhibition of the arginase enzyme from Leishmania amazonensis. Higher arginase inhibition was obsd. with fisetin, which was 4- and 10-times greater than the inhibition by quercetin and luteolin, resp. The hydroxyl group at position 3 may contribute to the inhibitory activity toward arginase, while the hydroxyl group at position 5 may not. The absence of the catechol group on apigenin drastically decreased the arginase inhibition. The docking of compds. showed that the inhibitors interacted with amino acids involved in the Mn2+-Mn2+ metal bridge formation at the catalytic site. Due to the low IC50 values of these flavonoids, they may be used as food supplements in leishmaniasis treatment.
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25. 25
  Mamani-Matsuda, M.; Rambert, J.; Malvy, D.; Lejoly-Boisseau, H.; Daulouède, S.; Thiolat, D.; Coves, S.; Courtois, P.; Vincendeau, P.; Mossalayi, M. D. Quercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophages Antimicrob. Agents Chemother. 2004, 48 (3) 924– 929 DOI: 10.1128/AAC.48.3.924-929.2004
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  25
  Quercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophages
  Mamani-Matsuda, Maria; Rambert, Jerome; Malvy, Denis; Lejoly-Boisseau, Helene; Daulouede, Sylvie; Thiolat, Denis; Coves, Sara; Courtois, Pierrette; Vincendeau, Philippe; Mossalayi, M. Djavad
  Antimicrobial Agents and Chemotherapy (2004), 48 (3), 924-929CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)
  In addn. to parasite spread, the severity of disease obsd. in cases of human African trypanosomiasis (HAT), or sleeping sickness, is assocd. with increased levels of inflammatory mediators, including tumor necrosis factor (TNF)-α and nitric oxide derivs. In the present study, quercetin (3,3',4',5,7-pentahydroxyflavone), a potent immunomodulating flavonoid, was shown to directly induce the death of Trypanosoma brucei gambiense, the causative agent of HAT, without affecting normal human cell viability. Quercetin directly promoted T. b. gambiense death by apoptosis as shown by Annexin V binding. In addn. to microbicidal activity, quercetin induced dose-dependent decreases in the levels of TNF-α and nitric oxide produced by activated human macrophages. These results highlight the potential use of quercetin as an antimicrobial and anti-inflammatory agent for the treatment of African trypanosomiasis.
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26. 26
  Dias, T. A.; Duarte, C. L.; Lima, C. F.; Proença, M. F.; Pereira-Wilson, C. Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin Eur. J. Med. Chem. 2013, 65, 500– 510 DOI: 10.1016/j.ejmech.2013.04.064
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  26
  Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin
  Dias, Tatiana A.; Duarte, Cecilia L.; Lima, Cristovao F.; Proenca, M. Fernanda; Pereira-Wilson, Cristina
  European Journal of Medicinal Chemistry (2013), 65 (), 500-510CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
  A series of chalcones I [R1 = H, HO, MeO; R2 = H, Me, MeO, Br; R3 = H, Me, MeO, etc.; R4, R5 = H, MeO] and flavonols II [R1 = H, MeO; R2 = H, Me, MeO, Br; R3 = H, Me, MeO, etc.; R4 = H, MeO] were synthesized in good yields by an eco-friendly approach. A pharmacol. evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed that the anticancer activity of flavonols was higher when compared with that of the resp. chalcone precursors. The antiproliferative activity of halogenated derivs. increases as the substituent in the 3- or 4-position of the B-ring goes from F to Cl and to Br. In addn., halogens in position 3 enhance anticancer activity in chalcones whereas for flavonol derivs. the best performance was registered for the 4-substituted derivs. Flow cytometry anal. showed that 2 compds. induced cell cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were corroborated by western blot and fluorescence microscopy anal. In summary, halogenated chalcones and flavonols were successfully prepd. and presented high anticancer activity as shown by their cell growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a pos. control.
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27. 27
  Juvale, K.; Stefan, K.; Wiese, M. Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2 Eur. J. Med. Chem. 2013, 67, 115– 126 DOI: 10.1016/j.ejmech.2013.06.035
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  27
  Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2
  Juvale, Kapil; Stefan, Katja; Wiese, Michael
  European Journal of Medicinal Chemistry (2013), 67 (), 115-126CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
  Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting mols. with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a soln. to overcome MDR. The authors synthesized a series of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 3' and 4' of the (benzo)flavone structure. All synthesized compds. were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compds. were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-assocd. protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addn. most active compds. were investigated for their cytotoxicity. It was obsd. that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH3 substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3',4'-OCH3 on Ph ring lead to increase in activity as compared to other substituents. Compd. 24, a 7,8-benzoflavone deriv. was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concns.
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28. 28
  Setzer, W. N.; Ogungbe, I. V. In-silico Investigation of Antitrypanosomal Phytochemicals from Nigerian Medicinal Plants PLoS Neglected Trop. Dis. 2012, 6 (7) e1727 DOI: 10.1371/journal.pntd.0001727
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  28
  In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plants
  Setzer, William N.; Ogungbe, Ifedayo V.
  PLoS Neglected Tropical Diseases (2012), 6 (7), e1727CODEN: PNTDAM; ISSN:1935-2735. (Public Library of Science)
  Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment. A mol. docking study has been carried out on phytochem. agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacol. to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compds. from 19 species of medicinal plants were investigated using in-silico mol. docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4' epimerase (TbUDPGE), and ornithine decarboxylase (TbODC). This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4' epimerase (TbUDPGE). Polyphenolic compds. such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins. This in-silico mol. docking study has identified potential biomol. targets of phytochem. components of antitrypanosomal plants and has detd. which phytochem. classes and structural manifolds likely target trypanosomal enzymes. The results could provide the framework for synthetic modification of bioactive phytochems., de novo synthesis of structural motifs, and lead to further phytochem. investigations.
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29. 29
  Kavanagh, K. L.; Jornvall, H.; Persson, B.; Oppermann, U. Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes Cell. Mol. Life Sci. 2008, 65, 3895– 3906 DOI: 10.1007/s00018-008-8588-y
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  29
  The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes
  Kavanagh, K. L.; Joernvall, H.; Persson, B.; Oppermann, U.
  Cellular and Molecular Life Sciences (2008), 65 (24), 3895-3906CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
  A review. Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have crit. roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metab. as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an α/β folding pattern with a central beta sheet flanked by 2-3 α-helixes from each side, thus a classical Rossmann fold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is detd. by a variable C-terminal segment. Relationships exist with bacterial haloalc. dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signaling.
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  Gourley, D. G.; Schuttelkopf, 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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  30
  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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  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 (6) 1457– 1468 DOI: 10.1111/j.1365-2958.2006.05332.x
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  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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  Chatelain, E. Chagas disease drug discovery: toward a new era J. Biomol. Screening 2015, 20 (1) 22– 35 DOI: 10.1177/1087057114550585
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  Chagas disease drug discovery: toward a new era
  Chatelain, Eric
  Journal of Biomolecular Screening (2015), 20 (1), 22-35, 14 pp.CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
  A review. American trypanosomiasis, or Chagas disease, is the result of infection by the Trypanosoma cruzi parasite. Endemic in Latin America where it is the major cause of death from cardiomyopathy, the impact of the disease is reaching global proportions through migrating populations. New drugs that are safe, efficacious, low cost, and adapted to the field are critically needed. Over the past five years, there has been increased interest in the disease and a surge in activities within various organizations. However, recent clin. trials with azoles, specifically posaconazole and the ravuconazole prodrug E1224, were disappointing, with treatment failure in Chagas patients reaching 70% to 90%, as opposed to 6% to 30% failure for benznidazole-treated patients. The lack of translation from in vitro and in vivo models to the clinic obsd. for the azoles raises several questions. There is a scientific requirement to review and challenge whether we are indeed using the right tools and decision-making processes to progress compds. forward for the treatment of this disease. New developments in the Chagas field, including new technologies and tools now available, will be discussed, and a redesign of the current screening strategy during the discovery process is proposed.
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  Katsuno, K.; Burrows, J. N.; Duncan, K.; Hooft van Huijsduijnen, R.; Kaneko, T.; Kita, K.; Mowbray, C. E.; Schmatz, D.; Warner, P.; Slingsby, B. T. Hit and lead criteria in drug discovery for infectious diseases of the developing world Nat. Rev. Drug Discovery 2015, 14 (11) 751– 758 DOI: 10.1038/nrd4683
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  Hit and lead criteria in drug discovery for infectious diseases of the developing world
  Katsuno, Kei; Burrows, Jeremy N.; Duncan, Ken; van Huijsduijnen, Rob Hooft; Kaneko, Takushi; Kita, Kiyoshi; Mowbray, Charles E.; Schmatz, Dennis; Warner, Peter; Slingsby, B. T.
  Nature Reviews Drug Discovery (2015), 14 (11), 751-758CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
  Reducing the burden of infectious diseases that affect people in the developing world requires sustained collaborative drug discovery efforts. The quality of the chem. starting points for such projects is a key factor in improving the likelihood of clin. success, and so it is important to set clear go/no-go criteria for the progression of hit and lead compds. With this in mind, the Japanese Global Health Innovative Technol. (GHIT) Fund convened with experts from the Medicines for Malaria Venture, the Drugs for Neglected Diseases initiative and the TB Alliance, together with representatives from the Bill & Melinda Gates Foundation, to set disease-specific criteria for hits and leads for malaria, tuberculosis, visceral leishmaniasis and Chagas disease. Here, we present the agreed criteria and discuss the underlying rationale.
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  Chou, T. C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies Pharmacol. Rev. 2006, 58 (3) 621– 681 DOI: 10.1124/pr.58.3.10
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  Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies
  Chou, Ting-Chao
  Pharmacological Reviews (2006), 58 (3), 621-681CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
  A review. The median-effect equation derived from the mass-action law principle at equil. steady state via math. induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theor. basis for the combination index (CI)-isobologram equation that allows quant. detn. of drug interactions, where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, resp. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-redn. index plot, and polygonogram for in vitro or in vivo studies. This theor. development, exptl. design, and computerized data anal. have facilitated dose-effect anal. for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of exptl. designs and real data anal. are also illustrated. The merging of the mass-action law principle with math. induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compd. or entity to how to beneficially use multiple drugs or modalities in combination therapies.
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35. 35
  Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation J. Mol. Biol. 1995, 245, 43– 53 DOI: 10.1016/S0022-2836(95)80037-9
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  Molecular recognition of a receptor sites using a genetic algorithm with a description of desolvation
  Jones, Gareth; Willett, Peter; Glen, Robert C.
  Journal of Molecular Biology (1995), 245 (1), 43-53CODEN: JMOBAK; ISSN:0022-2836. (Academic)
  Understanding the principles whereby macromol. biol. receptors can recognize small mol. substrates or inhibitors is the subject of a major effort. This is of paramount importance in rational drug design where the receptor structure is known (the "docking" problem). Current theor. approaches utilize models of the steric and electrostatic interaction of bound ligands and recently conformational flexibility has been incorporated. The authors report results based on software using a genetic algorithm that uses an evolutionary strategy in exploring the full conformational flexibility of the ligand with partial flexibility of the protein, and which satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Results are reported on five test systems showing excellent agreement with exptl. data. The design of the algorithm offers insight into the mol. recognition mechanism.
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36. 36
  Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking J. Mol. Biol. 1997, 267, 727– 748 DOI: 10.1006/jmbi.1996.0897
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  Development and validation of a genetic algorithm for flexible docking
  Jones, Gareth; Willett, Peter; Glen, Robert C.; Leach, Andrew R.; Taylor, Robin
  Journal of Molecular Biology (1997), 267 (3), 727-748CODEN: JMOBAK; ISSN:0022-2836. (Academic)
  Prediction of small mol. binding modes to macromols. of known three-dimensional structure is a problem of paramount importance in rational drug design (the "docking" problem). We report the development and validation of the program GOLD (Genetic Optimization for Ligand Docking). GOLD is an automated ligand docking program that uses a genetic algorithm to explore the full range of ligand conformational flexibility with partial flexibility of the protein and satisfies the fundamental requirement that the ligand must displace loosely bound water on binding. Numerous enhancements and modifications have been applied to the original technique resulting in a substantial increase in the reliability and the applicability of the algorithm. The advanced algorithm has been tested on a dataset of 100 complexes extd. from the Brookhaven Protein Data Bank. When used to dock the ligand back into the binding site, GOLD achieved a 71% success rate in identifying the exptl. binding mode.
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37. 37
  Schrödinger Release 2015-4: Maestro, version 10.4; Schrödinger, LLC: New York, 2015.
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38. 38
  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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39. 39
  Small-Molecule Drug Discovery Suite 2015-4: Glide, version 6.9; Schrödinger, LLC: New York, 2015.
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  There is no corresponding record for this reference.
40. 40
  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 Enclosure for Protein-Ligand Complexes J. Med. Chem. 2006, 49, 6177– 6196 DOI: 10.1021/jm051256o
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  40
  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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41. 41
  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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42. 42
  Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shaw, D. E.; Shelley, M.; Perry, J. K.; 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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43. 43
  Li, H.; Robertson, A. D.; Jensen, J. H. Very Fast Empirical Prediction and Interpretation of Protein pKa Values Proteins: Struct., Funct., Genet. 2005, 61, 704– 721 DOI: 10.1002/prot.20660
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  Very fast empirical prediction and rationalization of protein pKa values
  Li, Hui; Robertson, Andrew D.; Jensen, Jan H.
  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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44. 44
  Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, O. M. H.; 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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45. 45
  Benvenuti, M.; Mangani, S. Crystallization of soluble proteins in vapor diffusion for x-ray crystallography Nat. Protoc. 2007, 2 (7) 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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46. 46
  Leslie, A. G. The integration of macromolecular diffraction data Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48– 57 DOI: 10.1107/S0907444905039107
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  The integration of macromolecular diffraction data
  Leslie, Andrew G. W.
  Acta Crystallographica, Section D: Biological Crystallography (2006), D62 (1), 48-57CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
  The objective of any modern data-processing program is to produce from a set of diffraction images a set of indexes (hkls) with their assocd. intensities (and ests. of their uncertainties), together with an accurate est. of the crystal unit-cell parameters. This procedure should not only be reliable, but should involve an abs. min. of user intervention. The process can be conveniently divided into three stages. The first (autoindexing) dets. the unit-cell parameters and the orientation of the crystal. The unit-cell parameters may indicate the likely Laue group of the crystal. The second step is to refine the initial est. of the unit-cell parameters and also the crystal mosaicity using a procedure known as post-refinement. The third step is to integrate the images, which consists of predicting the positions of the Bragg reflections on each image and obtaining an est. of the intensity of each reflection and its uncertainty. This is carried out while simultaneously refining various detector and crystal parameters. Basic features of the algorithms employed for each of these three sep. steps are described, principally with ref. to the program MOSFLM.
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  Evans, P. Scaling and assessment of data quality Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72– 82 DOI: 10.1107/S0907444905036693
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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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  Journal of Applied Crystallography (1997), 30 (6), 1022-1025CODEN: JACGAR; ISSN:0021-8898. (Munksgaard International Publishers Ltd.)
  MOLREP is an automated program for mol. replacement which uses effective new approaches in data processing and rotational and translational searching. These include an automatic choice of all parameters, scaling by Patterson origin peaks and soft resoln. cutoff. One of the cornerstones of the program is an original full-symmetry translation function combined with a packing function. Information from the model already placed in the cell is incorporated in both translation and packing functions. A no. of tests using exptl. data proved the ability of the program to find the correct soln. in difficult cases.
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  Dawson, A.; Tulloch, L. B.; Barrack, K. L.; Hunter, W. N. High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 1334– 1340 DOI: 10.1107/S0907444910040886
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  High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target
  Dawson, Alice; Tulloch, Lindsay B.; Barrack, Keri L.; Hunter, William N.
  Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (12), 1334-1340CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
  Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resoln. structures have been detd., using data between 1.6 and 1.1 Å resoln., of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine deriv. The structures provide insight into the interactions formed by new mol. entities in the enzyme active site with ligands that represent lead compds. for structure-based inhibitor development and to support early-stage drug discovery.
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  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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  The PROCHECK suite of programs provides a detailed check on the stereochem. of a protein structure. Its outputs comprise a no. of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well-refined structures of the same resoln. and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modeled on known structures.
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  McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E. M. Presenting your structures: the CCP4mg molecular-graphics software Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 386– 394 DOI: 10.1107/S0907444911007281
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  McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E. M.
  Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 386-394CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
  CCP4mg is a mol.-graphics program that is designed to give rapid access to both straightforward and complex static and dynamic representations of macromol. structures. It has recently been updated with a new interface that provides more sophisticated atom-selection options and a wizard to facilitate the generation of complex scenes. These scenes may contain a mixt. of coordinate-derived and abstr. graphical objects, including text objects, arbitrary vectors, geometric objects and imported images, which can enhance a picture and eliminate the need for subsequent editing. Scene descriptions can be saved to file and transferred to other mols. Here, the substantially enhanced version 2 of the program, with a new underlying GUI toolkit, is described. A built-in rendering module produces publication-quality images.
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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 (2) 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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  Sereno, D.; Cavaleyra, M.; Zemzoumi, K.; Maquaire, S.; Ouaissi, A.; Lemesre, J. L. Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action Antimicrob. Agents Chemother. 1998, 42 (12) 3097– 3102
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  Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action
  Sereno D; Cavaleyra M; Zemzoumi K; Maquaire S; Ouaissi A; Lemesre J L
  Antimicrobial agents and chemotherapy (1998), 42 (12), 3097-102 ISSN:0066-4804.
  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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57. 57
  Bowling, T.; Mercer, L.; Don, R.; Jacobs, R.; Nare, B. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis Int. J. Parasitol.: Drugs Drug Resist. 2012, 2, 262– 270 DOI: 10.1016/j.ijpddr.2012.02.002
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  Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis
  Bowling Tana; Mercer Luke; Jacobs Robert; Nare Bakela; Don Robert
  International journal for parasitology. Drugs and drug resistance (2012), 2 (), 262-70 ISSN:2211-3207.
  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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58. 58
  Bifeld, E.; Tejera Nevado, P.; Bartsch, J.; Eick, J.; Clos, J. A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues. Med. Microbiol. Immunol. 2016, epub ahead of print.
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  Shia, C.; Tsai, S.; Kuo, S.; Hou, Y.; Chao, P. L. Metabolism and pharmacokinetics of 3,3‘,4‘,7- tetrahydeoxyflavone (fisetin), 5-hydroxyflavone and 7-hydroxyflavone and antihemolysis effects of fisetin and its serum metabolites J. Agric. Food Chem. 2009, 57, 83– 89 DOI: 10.1021/jf802378q
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  Metabolism and Pharmacokinetics of 3,3',4',7-Tetrahydroxyflavone (Fisetin), 5-Hydroxyflavone, and 7-Hydroxyflavone and Antihemolysis Effects of Fisetin and Its Serum Metabolites
  Shia, Chi-Sheng; Tsai, Shang-Yuan; Kuo, Sheng-Chu; Hou, Yu-Chi; Chao, Pei-Dawn Lee
  Journal of Agricultural and Food Chemistry (2009), 57 (1), 83-89CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
  3,3',4',7-Tetrahydroxyflavone (fisetin) has shown various beneficial bioactivities. This study investigated the metab. and pharmacokinetics of fisetin, 5-hydroxyflavone (5-OH-flavone), and 7-hydroxyflavone (7-OH-flavone) in male Sprague-Dawley rats. Blood was withdrawn via cardiopuncture and assayed by HPLC before and after hydrolysis with sulfatase and β-glucuronidase. The results indicated that after i.v. administration of fisetin (10 mg/kg of bw), fisetin declined rapidly and fisetin sulfates/glucuronides emerged instantaneously. When fisetin (50 mg/kg of bw) was given orally, fisetin parent form was transiently present in serum only during the absorption phase, whereas fisetin sulfates/glucuronides predominated. The serum metabolites of fisetin showed less potent inhibition on 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced hemolysis than fisetin. Following oral administrations of 40 mg/kg of bw of 5-OH-flavone and 7-OH-flavone, the glucuronide of 5-OH-flavone and the sulfate/glucuronide of 7-OH-flavone were found in serum, whereas no traces of parent forms were detected. In conclusion, fisetin and 7-OH-flavone were rapidly and extensively biotransformed into their sulfate/glucuronide, whereas 5-OH-flavone was exclusively metabolized to glucuronide.
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Supporting Information
Supporting Information
ARTICLE SECTIONS
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00698.
- Compound characterization checklist (XLS)
- Molecular formula strings (XLSX)
- Compound 1–TbPTR1 (PDB)
- Compound 2–TbPTR1 (PDB)
- Compound 3–TbPTR1 (PDB)
- Compound 4–TbPTR1 (PDB)
- Compound 5–TbPTR1 (PDB)
- Compound 6–TbPTR1 (PDB)
- Compound 7–TbPTR1 (PDB)
- Compound 8–TbPTR1 (PDB)
- Compound 9–TbPTR1 (PDB)
- Compound 10–TbPTR1 (PDB)
- Compound 11–TbPTR1 (PDB)
- Compound 12–TbPTR1 (PDB)
- Compound 13–TbPTR1 (PDB)
- Compound 14–TbPTR1 (PDB)
- Compound 15–TbPTR1 (PDB)
- Compound 16–TbPTR1 (PDB)
- Compound 1–LmPTR1 (PDB)
- Compound 2–LmPTR1 (PDB)
- Compound 3–LmPTR1 (PDB)
- Compound 4–LmPTR1 (PDB)
- Compound 5–LmPTR1 (PDB)
- Compound 6–LmPTR1 (PDB)
- Compound 7–LmPTR1 (PDB)
- Compound 8–LmPTR1 (PDB)
- Compound 9–LmPTR1 (PDB)
- Compound 10–LmPTR1 (PDB)
- Compound 11–LmPTR1 (PDB)
- Compound 12–LmPTR1 (PDB)
- Compound 13–LmPTR1 (PDB)
- Compound 14–LmPTR1 (PDB)
- Compound 15–LmPTR1 (PDB)
- Compound 16–LmPTR1 (PDB)
- General structures of the flavonoids; code, chemical structure, purity, and IC₅₀ values of the 38 natural compounds screened; X-ray crystallographic data; inhibitory activity of the synthetic flavonols against TbPTR1 and LmPTR1; docking analysis; sequence alignment of TbPTR1 and LmPTR1; inhibitory activity of the synthetic flavonols against hDHFR, TbDHFR and LmDHFR; ADME-Tox data; antiparasitic activity of the synthesized compounds alone and in combination with MTX and synergy coefficients; isobologram and dose–effect curves for the combination of MTX and compound 13; toxicity of the combination on THP-1 cells; plasma concentration of compound 2 in BALB/c mice; nanoparticles characterization by dynamic light scattering; crystal structures used for conserved water analysis in TbPTR1 and LmPTR1; kinetic characterization of PTR1 and DHFR; characterization of the compounds 1–16 and of the intermediates 17–24 (PDF)
Accession Codes
PDB code 5JCJ was used for docking of compounds 1–16 (TbPTR1). PDB code 1E92 was used for docking of compounds 1–16 (LmPTR1). The Protein Data Bank accession codes of the X-ray crystallographic structures of TbPTR1 in complex with NP-13, NP-29, 2, and 7 are 5JDC, 5JCX, 5JDI, and 5JCJ, respectively. Authors will release the atomic coordinates and experimental data upon article publication.

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Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs

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Abstract

Introduction

Results and Discussion

On-Target Screening of a Natural Product Library and Hit Identification

Figure 1

X-ray Crystallographic Studies of the Natural Products

TbPTR1-NADPH/NADP+-NP-29

Figure 2

TbPTR1-NADPH/NADP+-NP-13

Flavonoid Library Design and Synthesis

Figure 3

Scheme 1

Target Compound Profile

Testing of Synthetic Flavonols against PTR1 Enzymes

Figure 4

Crystal Structures of Synthetic Flavonoid–TbPTR1 Complexes

TbPTR1-NADPH/NADP+–2

TbPTR1-NADPH/NADP+–7

Figure 5

Computational Docking of Synthetic Compounds to TbPTR1 and LmPTR1 and SAR Analysis

Figure 6

Figure 7

Testing of Synthetic Flavonols against TbDHFR, LmDHFR, hDHFR, and hTS

Early Toxicity Studies

Figure 8

Testing of Synthetic Compounds on Cultured Parasites

Figure 9

Combination Studies and Evaluation of Synergy

Figure 10

Pharmacokinetic Studies, in Vivo Assays, and Compound Delivery

Conclusion

Experimental Section

Preparation of the Natural Products Library

Computational Studies

Virtual Screening of the Natural Product Library

Docking of Synthetic Flavonoid Derivatives

Synthesis

General

General Procedure for the Synthesis of (2E)-1-(2-Hydroxy-phenyl)-3-phenylprop-2-en-1-ones

General Procedure for the Synthesis of Methylated Flavonols (Compounds 9–16)

General Procedure for the Synthesis of Demethylated Flavonols (Compounds 1–8)

Protein Expression and Purification

X-ray Crystallography

TbPTR1, LmPTR1, TbDHFR, LmDHFR, hTS, and hDHFR Target Enzyme Assays

In Vitro Biological Assays

In Vitro Evaluation of Activity against L. infantum Intramacrophage Amastigotes

In Vitro Evaluation of Activity against T. brucei

In Vitro Evaluation of Activity against T. cruzi

Cytotoxicity Assessment against THP-1 Macrophages

hERG Cardiotoxicity Assay

Cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 Assay

Cytotoxicity Assay against A549 and WI38 Cells

Assessment of Mitochondrial Toxicity

Aurora B Kinase Assay

Solubility Assays

Compound 2 Encapsulation in PLGA and Solubilization with Cyclodextrins

Pharmacokinetics of Compound 2

Ethics Statement

Supporting Information

Terms & Conditions

Author Information

Acknowledgment

Abbreviations Used

References

Cited By

Abstract

Figure 1

Figure 2

Figure 3

Scheme 1

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

TbPTR1-NADPH/NADP⁺-NP-29

TbPTR1-NADPH/NADP⁺-NP-13

TbPTR1-NADPH/NADP⁺–2

TbPTR1-NADPH/NADP⁺–7