Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs
- Chiara Borsari
- ,
- Rosaria Luciani
- ,
- Cecilia Pozzi
- ,
- Ina Poehner
- ,
- Stefan Henrich
- ,
- Matteo Trande
- ,
- Anabela Cordeiro-da-Silva
- ,
- Nuno Santarem
- ,
- Catarina Baptista
- ,
- Annalisa Tait
- ,
- Flavio Di Pisa
- ,
- Lucia Dello Iacono
- ,
- Giacomo Landi
- ,
- Sheraz Gul
- ,
- Markus Wolf
- ,
- Maria Kuzikov
- ,
- Bernhard Ellinger
- ,
- Jeanette Reinshagen
- ,
- Gesa Witt
- ,
- Philip Gribbon
- ,
- Manfred Kohler
- ,
- Oliver Keminer
- ,
- Birte Behrens
- ,
- Luca Costantino
- ,
- Paloma Tejera Nevado
- ,
- Eugenia Bifeld
- ,
- Julia Eick
- ,
- Joachim Clos
- ,
- Juan Torrado
- ,
- María D. Jiménez-Antón
- ,
- María J. Corral
- ,
- José Ma Alunda
- ,
- Federica Pellati
- ,
- Rebecca C. Wade
- ,
- Stefania Ferrari
- ,
- Stefano Mangani
- , and
- Maria Paola Costi
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
Results and Discussion
On-Target Screening of a Natural Product Library and Hit Identification
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.
X-ray Crystallographic Studies of the Natural Products
TbPTR1-NADPH/NADP+-NP-29
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 2Fo – Fc 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
Flavonoid Library Design and Synthesis

compd | R5 | R7 | R2′ | R3′ | R4′ | 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

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.
Scheme 1

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.
Target Compound Profile
Testing of Synthetic Flavonols against PTR1 Enzymes
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).
Crystal Structures of Synthetic Flavonoid–TbPTR1 Complexes
TbPTR1-NADPH/NADP+–2
TbPTR1-NADPH/NADP+–7
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.
Computational Docking of Synthetic Compounds to TbPTR1 and LmPTR1 and SAR Analysis
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.
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.
Testing of Synthetic Flavonols against TbDHFR, LmDHFR, hDHFR, and hTS
Early Toxicity Studies
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.
Testing of Synthetic Compounds on Cultured Parasites
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.
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.
Combination Studies and Evaluation of Synergy
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.
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 ± 3.71b | 41.95 ± 10.52b | 127.36 ± 60.20b | 278.17 ± 172.13b |
11.72 ± 2.79b | 27.45 ± 6.90b | 65.56 ± 16.42b | 119.72 ± 29.15b |
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
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
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
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)
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.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
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.
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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- 4Barrett, 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.11.88Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MfnsVGktA%253D%253D&md5=2312d9bfca4c877e3401fb2ec8a3875dDrug resistance in human African trypanosomiasisBarrett Michael P; Vincent Isabel M; Burchmore Richard J S; Kazibwe Anne J N; Matovu EnockFuture 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.
- 5Chakravarty, J.; Sundar, S. Drug resistance in Leishmaniasis J. Glob. Infect. Dis. 2010, 2, 167– 176 DOI: 10.4103/0974-777X.62887Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3cnlt1SgsA%253D%253D&md5=ba84dd789e799866f6a315d9ce962fcfDrug resistance in leishmaniasisChakravarty Jaya; Sundar ShyamJournal 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.
- 6Chatelain, 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.S16381Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3MvptlKisQ%253D%253D&md5=23c22bc704d2babf753052ec470a75b0Drug discovery and development for neglected diseases: the DNDi modelChatelain Eric; Ioset Jean-RobertDrug 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.
- 7Gilbert, I. H. Drug discovery for neglected diseases: molecular target-based and phenotypic approaches J. Med. Chem. 2013, 56 (20) 7719– 7726 DOI: 10.1021/jm400362bGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVWkur%252FF&md5=dcc54f2ed7987c3089bbe4944b747420Drug Discovery for Neglected Diseases: Molecular Target-Based and Phenotypic ApproachesGilbert, 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.
- 8Hyde, J. E. Exploring the folate pathway in Plasmodium falciparum Acta Trop. 2005, 94 (3) 191– 206 DOI: 10.1016/j.actatropica.2005.04.002Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXks1agtL0%253D&md5=4a5d27c5bae52f2f32e66aff4293ba37Exploring the folate pathway in Plasmodium falciparumHyde, 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.
- 9Gilbert, 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)00088-1Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XkslKjurc%253D&md5=98b5492137fa17801f6a8808e5e50624Inhibitors of dihydrofolate reductase in leishmania and trypanosomesGilbert, 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.
- 10Dawson, 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.xGoogle Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFensbrF&md5=aa70764962fef14898e2b1bb22e646b3Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexateDawson, 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.
- 11Guerrieri, 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.2012.11.013Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXislyrtr0%253D&md5=8be34fc5791f1d2b915ab598fa852a71Biochemical effects of riluzole on Leishmania parasitesGuerrieri, 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.
- 12Barrack, 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/S174430911004724XGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1yntQ%253D%253D&md5=9a59801fee685e600875de86ad2b6127Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active siteBarrack, 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.
- 13Ferrari, 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/jm1010572Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFShtbvM&md5=1e0515cb0a3d0717094880b98f8ba6e6Virtual Screening Identification of Nonfolate Compounds, Including a CNS Drug, as Antiparasitic Agents Inhibiting Pteridine ReductaseFerrari, 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. PaolaJournal 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.
- 14Tulloch, 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/jm901059xGoogle Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWrtb%252FP&md5=05b3c63323eac4e39a7491a0bfd60fb2Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the LeishmaniasisTulloch, 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.
- 15Cavazzuti, 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.0704384105Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhvFCitrk%253D&md5=d2477ec35df976858f13b6d14d283cadDiscovery of potent pteridine reductase inhibitors to guide antiparasite drug developmentCavazzuti, 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 PaolaProceedings 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.
- 16Balunas, M. J.; Kinghorn, A. D. Drug discovery from medicinal plants Life Sci. 2005, 78 (5) 431– 441 DOI: 10.1016/j.lfs.2005.09.012Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1ejsrbP&md5=a73341f37453a6d61eee734d0cb27a22Drug discovery from medicinal plantsBalunas, Marcy J.; Kinghorn, A. DouglasLife 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.
- 17Annang, 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: 10.1177/1087057114555846Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlGjsA%253D%253D&md5=161347b921b221a4712a1aa5d429cc7aHigh-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human african trypanosomiasis, leishmaniasis, and chagas diseaseAnnang, 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. GonzalezJournal 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.
- 18Harvey, A. L. Natural products in drug discovery Drug Discovery Today 2008, 13 (19–20) 894– 901 DOI: 10.1016/j.drudis.2008.07.004Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFCgu7fJ&md5=da90f7cf60b799a14b6231f85b4eaf47Natural products in drug discoveryHarvey, 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.
- 19Harvey, 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/nrd4510Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVarsbg%253D&md5=b871148315dc0623683498fa15205b14The re-emergence of natural products for drug discovery in the genomics eraHarvey, 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.
- 20Ndjonka, 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/ijms14023395Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtVKis70%253D&md5=fc2ee3e0dfda6e3983cc808a8033b35dNatural products as a source for treating neglected parasitic diseasesNdjonka, Dieudonne; Rapado, Ludmila Nakamura; Silber, Ariel M.; Liebau, Eva; Wrenger, CarstenInternational 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.
- 21Singh, 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.2013.11.048Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOgtrjL&md5=d3f82091638d595b388c6f65080ce8d9Natural product based leads to fight against leishmaniasisSingh, 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.
- 22Tasdemir, 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.50.4.1352-1364.2006Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFOrs74%253D&md5=b619033c75dc319f38f41a8308dbd77aAntitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studiesTasdemir, Deniz; Kaiser, Marcel; Brun, Reto; Yardley, Vanessa; Schmidt, Thomas J.; Tosun, Fatma; Ruedi, PeterAntimicrobial 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.
- 23da 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.2012.01.015Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivV2hsLw%253D&md5=cefb6be8bd71da4f18a7c0f1036f2723The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginaseda Silva, Edson Roberto; Maquiaveli, Claudia do Carmo; Magalhaes, Prislaine PupolinExperimental 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.
- 24Manjolin, 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.2013.05.025Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFWkurnP&md5=cdab0fb1292fc6c7e9b937fef699dbb1Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infectionManjolin, Leticia Correa; dos Reis, Matheus Balduino Goncalves; Maquiaveli, Claudia do Carmo; Santos-Filho, Osvaldo Andrade; da Silva, Edson RobertoFood 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.
- 25Mamani-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.48.3.924-929.2004Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXitVGht7Y%253D&md5=ac73c2ff4fa9568cfa778d33418b1dfbQuercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophagesMamani-Matsuda, Maria; Rambert, Jerome; Malvy, Denis; Lejoly-Boisseau, Helene; Daulouede, Sylvie; Thiolat, Denis; Coves, Sara; Courtois, Pierrette; Vincendeau, Philippe; Mossalayi, M. DjavadAntimicrobial 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.
- 26Dias, 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.2013.04.064Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFemurvE&md5=d73c3f07d3fadc45550fd9bc6e671f63Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetinDias, Tatiana A.; Duarte, Cecilia L.; Lima, Cristovao F.; Proenca, M. Fernanda; Pereira-Wilson, CristinaEuropean 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.
- 27Juvale, 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.2013.06.035Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVeis73E&md5=961e1899a816717d108c5c8e71944f70Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2Juvale, Kapil; Stefan, Katja; Wiese, MichaelEuropean 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.
- 28Setzer, 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.0001727Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFWlsrvO&md5=a733aa0446bda89a0ad5775e6ac28d93In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plantsSetzer, 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.
- 29Kavanagh, 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-yGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFCgs7nO&md5=cc60414b9811fc9ab32f6ee7cc8ef3d5The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymesKavanagh, 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.
- 30Gourley, 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: 10.1038/88584Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktFektLw%253D&md5=b5be3a57375838e0a9b41429794ba37fPteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasitesGourley, 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.
- 31Dawson, 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.xGoogle Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFensbrF&md5=aa70764962fef14898e2b1bb22e646b3Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexateDawson, 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.
- 32Chatelain, E. Chagas disease drug discovery: toward a new era J. Biomol. Screening 2015, 20 (1) 22– 35 DOI: 10.1177/1087057114550585Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlKisg%253D%253D&md5=dbdc4ccf1c61a130e7e8ed6c1ed08e71Chagas disease drug discovery: toward a new eraChatelain, EricJournal 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.
- 33Katsuno, 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/nrd4683Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1SitL7O&md5=6cd6d7c8ca3a85d64f24915da8e1120cHit and lead criteria in drug discovery for infectious diseases of the developing worldKatsuno, 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.
- 34Chou, 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.58.3.10Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVOhtLfL&md5=374c53e0772c679d1cdb08ff3112be90Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studiesChou, Ting-ChaoPharmacological 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.
- 35Jones, 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)80037-9Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjtVKlsbc%253D&md5=bf45edafb986a73c7c1d0ac55e070f28Molecular recognition of a receptor sites using a genetic algorithm with a description of desolvationJones, 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.
- 36Jones, 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.1996.0897Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXis1KntLo%253D&md5=476a2b1d8f80f3ba418052fe29d735caDevelopment and validation of a genetic algorithm for flexible dockingJones, Gareth; Willett, Peter; Glen, Robert C.; Leach, Andrew R.; Taylor, RobinJournal 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.
- 37Schrödinger Release 2015-4: Maestro, version 10.4; Schrödinger, LLC: New York, 2015.Google ScholarThere is no corresponding record for this reference.
- 38Sanschagrin, 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.5560071002Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntVSlsrY%253D&md5=eb46b53aee05dfec17053634a0ce2980Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificitySanschagrin, 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.
- 39Small-Molecule Drug Discovery Suite 2015-4: Glide, version 6.9; Schrödinger, LLC: New York, 2015.Google ScholarThere is no corresponding record for this reference.
- 40Friesner, 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/jm051256oGoogle Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVGmurg%253D&md5=ea428c82ead0d8c27f8c1a7b694a1edfExtra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand ComplexesFriesner, 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.
- 41Halgren, 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/jm030644sGoogle Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyit78%253D&md5=33d68dd968e65626b449df61e44e37beGlide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screeningHalgren, 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.
- 42Friesner, 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/jm0306430Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyit74%253D&md5=8cc2f0022318b12dd972e9c493375bf9Glide: A new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracyFriesner, 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.
- 43Li, 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.20660Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlaltLnN&md5=9e33529954c65867929326b99dac493bVery fast empirical prediction and rationalization of protein pKa valuesLi, 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.
- 44Cardinale, 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.1104829108Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFams7jM&md5=46b7c3dde0c96c0c3d053a58aa84856fProtein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthaseCardinale, 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. PaolaProceedings 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.
- 45Benvenuti, 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.2007.198Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFahurzF&md5=609fceaec4d8b9b3ad5a129f57989e68Crystallization of soluble proteins in vapor diffusion for X-ray crystallographyBenvenuti, Manuela; Mangani, StefanoNature 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.
- 46Leslie, A. G. The integration of macromolecular diffraction data Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48– 57 DOI: 10.1107/S0907444905039107Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7vP&md5=70d65687f3b3426c93e96bfb706e0c5cThe integration of macromolecular diffraction dataLeslie, 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.
- 47Evans, P. Scaling and assessment of data quality Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72– 82 DOI: 10.1107/S0907444905036693Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7jM&md5=293d3876e534c0c57813990515bb3c76Scaling and assessment of data qualityEvans, PhilipActa 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.
- 48Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760– 763. DOI: 10.1107/S0907444994003112Google ScholarThere is no corresponding record for this reference.
- 49Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement J. Appl. Crystallogr. 1997, 30, 1022– 1025 DOI: 10.1107/S0021889897006766Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhslehu7Y%253D&md5=96af64aedd8a8f018c3c18b57a6b4b21MOLREP: an automated program for molecular replacementVagin, Alexei; Teplyakov, AlexeiJournal 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.
- 50Dawson, 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/S0907444910040886Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFSgsr3O&md5=3d2805846022b2bf45531dd8f156786cHigh-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 targetDawson, 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.
- 51Murshudov, 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/S0907444996012255Google ScholarThere is no corresponding record for this reference.
- 52Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126– 2132 DOI: 10.1107/S0907444904019158Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVars73P&md5=1be390f3bb6fd584468499ad0921161eCoot: model-building tools for molecular graphicsEmsley, Paul; Cowtan, KevinActa 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'.
- 53Laskowski, 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/S0021889892009944Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXit12lurY%253D&md5=fb69fc4410cd716aaaa7cc0db06b3ed2PROCHECK: a program to check the stereochemical quality of protein structuresLaskowski, 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.
- 54McNicholas, 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/S0907444911007281Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqurw%253D&md5=d96403a39bc723a66f11a730f81f76c3Presenting your structures: The CCP4mg molecular-graphics softwareMcNicholas, 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.
- 55Shanks, 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.2009.09.003Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFShtb3N&md5=73a55624db0bf7bc04b768bef03807dfDevelopment and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductaseShanks, 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.
- 56Sereno, 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– 3102Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M%252FlsVynug%253D%253D&md5=6e1275a26903f7ffdc91c68a458b8d0fAxenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of actionSereno D; Cavaleyra M; Zemzoumi K; Maquaire S; Ouaissi A; Lemesre J LAntimicrobial 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.
- 57Bowling, 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.2012.02.002Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cvls1egsg%253D%253D&md5=a8e04cd8cedfe8b8f505ff704ecce376Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasisBowling Tana; Mercer Luke; Jacobs Robert; Nare Bakela; Don RobertInternational 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.
- 58Bifeld, 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.Google ScholarThere is no corresponding record for this reference.
- 59Shia, 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/jf802378qGoogle Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFWhsLzO&md5=111578f6be5b32f02343ccdc5a2509f2Metabolism and Pharmacokinetics of 3,3',4',7-Tetrahydroxyflavone (Fisetin), 5-Hydroxyflavone, and 7-Hydroxyflavone and Antihemolysis Effects of Fisetin and Its Serum MetabolitesShia, Chi-Sheng; Tsai, Shang-Yuan; Kuo, Sheng-Chu; Hou, Yu-Chi; Chao, Pei-Dawn LeeJournal 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
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.
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 2Fo – Fc 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.
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.
Scheme 1
Scheme 1. Synthesis of the Flavonols 1–16aScheme 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).
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.
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.
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.
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.
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.
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.
References
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- 8Hyde, J. E. Exploring the folate pathway in Plasmodium falciparum Acta Trop. 2005, 94 (3) 191– 206 DOI: 10.1016/j.actatropica.2005.04.002Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXks1agtL0%253D&md5=4a5d27c5bae52f2f32e66aff4293ba37Exploring the folate pathway in Plasmodium falciparumHyde, 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.
- 9Gilbert, 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)00088-1Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XkslKjurc%253D&md5=98b5492137fa17801f6a8808e5e50624Inhibitors of dihydrofolate reductase in leishmania and trypanosomesGilbert, 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.
- 10Dawson, 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.xGoogle Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFensbrF&md5=aa70764962fef14898e2b1bb22e646b3Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexateDawson, 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.
- 11Guerrieri, 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.2012.11.013Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXislyrtr0%253D&md5=8be34fc5791f1d2b915ab598fa852a71Biochemical effects of riluzole on Leishmania parasitesGuerrieri, 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.
- 12Barrack, 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/S174430911004724XGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1yntQ%253D%253D&md5=9a59801fee685e600875de86ad2b6127Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active siteBarrack, 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.
- 13Ferrari, 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/jm1010572Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFShtbvM&md5=1e0515cb0a3d0717094880b98f8ba6e6Virtual Screening Identification of Nonfolate Compounds, Including a CNS Drug, as Antiparasitic Agents Inhibiting Pteridine ReductaseFerrari, 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. PaolaJournal 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.
- 14Tulloch, 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/jm901059xGoogle Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWrtb%252FP&md5=05b3c63323eac4e39a7491a0bfd60fb2Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the LeishmaniasisTulloch, 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.
- 15Cavazzuti, 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.0704384105Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhvFCitrk%253D&md5=d2477ec35df976858f13b6d14d283cadDiscovery of potent pteridine reductase inhibitors to guide antiparasite drug developmentCavazzuti, 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 PaolaProceedings 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.
- 16Balunas, M. J.; Kinghorn, A. D. Drug discovery from medicinal plants Life Sci. 2005, 78 (5) 431– 441 DOI: 10.1016/j.lfs.2005.09.012Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1ejsrbP&md5=a73341f37453a6d61eee734d0cb27a22Drug discovery from medicinal plantsBalunas, Marcy J.; Kinghorn, A. DouglasLife 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.
- 17Annang, 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: 10.1177/1087057114555846Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlGjsA%253D%253D&md5=161347b921b221a4712a1aa5d429cc7aHigh-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human african trypanosomiasis, leishmaniasis, and chagas diseaseAnnang, 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. GonzalezJournal 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.
- 18Harvey, A. L. Natural products in drug discovery Drug Discovery Today 2008, 13 (19–20) 894– 901 DOI: 10.1016/j.drudis.2008.07.004Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtFCgu7fJ&md5=da90f7cf60b799a14b6231f85b4eaf47Natural products in drug discoveryHarvey, 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.
- 19Harvey, 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/nrd4510Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVarsbg%253D&md5=b871148315dc0623683498fa15205b14The re-emergence of natural products for drug discovery in the genomics eraHarvey, 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.
- 20Ndjonka, 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/ijms14023395Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjtVKis70%253D&md5=fc2ee3e0dfda6e3983cc808a8033b35dNatural products as a source for treating neglected parasitic diseasesNdjonka, Dieudonne; Rapado, Ludmila Nakamura; Silber, Ariel M.; Liebau, Eva; Wrenger, CarstenInternational 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.
- 21Singh, 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.2013.11.048Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOgtrjL&md5=d3f82091638d595b388c6f65080ce8d9Natural product based leads to fight against leishmaniasisSingh, 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.
- 22Tasdemir, 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.50.4.1352-1364.2006Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XjvFOrs74%253D&md5=b619033c75dc319f38f41a8308dbd77aAntitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studiesTasdemir, Deniz; Kaiser, Marcel; Brun, Reto; Yardley, Vanessa; Schmidt, Thomas J.; Tosun, Fatma; Ruedi, PeterAntimicrobial 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.
- 23da 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.2012.01.015Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XivV2hsLw%253D&md5=cefb6be8bd71da4f18a7c0f1036f2723The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginaseda Silva, Edson Roberto; Maquiaveli, Claudia do Carmo; Magalhaes, Prislaine PupolinExperimental 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.
- 24Manjolin, 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.2013.05.025Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFWkurnP&md5=cdab0fb1292fc6c7e9b937fef699dbb1Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infectionManjolin, Leticia Correa; dos Reis, Matheus Balduino Goncalves; Maquiaveli, Claudia do Carmo; Santos-Filho, Osvaldo Andrade; da Silva, Edson RobertoFood 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.
- 25Mamani-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.48.3.924-929.2004Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXitVGht7Y%253D&md5=ac73c2ff4fa9568cfa778d33418b1dfbQuercetin induces apoptosis of Trypanosoma brucei gambiense and decreases the proinflammatory response of human macrophagesMamani-Matsuda, Maria; Rambert, Jerome; Malvy, Denis; Lejoly-Boisseau, Helene; Daulouede, Sylvie; Thiolat, Denis; Coves, Sara; Courtois, Pierrette; Vincendeau, Philippe; Mossalayi, M. DjavadAntimicrobial 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.
- 26Dias, 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.2013.04.064Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFemurvE&md5=d73c3f07d3fadc45550fd9bc6e671f63Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetinDias, Tatiana A.; Duarte, Cecilia L.; Lima, Cristovao F.; Proenca, M. Fernanda; Pereira-Wilson, CristinaEuropean 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.
- 27Juvale, 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.2013.06.035Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVeis73E&md5=961e1899a816717d108c5c8e71944f70Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2Juvale, Kapil; Stefan, Katja; Wiese, MichaelEuropean 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.
- 28Setzer, 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.0001727Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFWlsrvO&md5=a733aa0446bda89a0ad5775e6ac28d93In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plantsSetzer, 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.
- 29Kavanagh, 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-yGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFCgs7nO&md5=cc60414b9811fc9ab32f6ee7cc8ef3d5The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymesKavanagh, 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.
- 30Gourley, 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: 10.1038/88584Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXktFektLw%253D&md5=b5be3a57375838e0a9b41429794ba37fPteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasitesGourley, 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.
- 31Dawson, 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.xGoogle Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFensbrF&md5=aa70764962fef14898e2b1bb22e646b3Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexateDawson, 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.
- 32Chatelain, E. Chagas disease drug discovery: toward a new era J. Biomol. Screening 2015, 20 (1) 22– 35 DOI: 10.1177/1087057114550585Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlKisg%253D%253D&md5=dbdc4ccf1c61a130e7e8ed6c1ed08e71Chagas disease drug discovery: toward a new eraChatelain, EricJournal 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.
- 33Katsuno, 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/nrd4683Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1SitL7O&md5=6cd6d7c8ca3a85d64f24915da8e1120cHit and lead criteria in drug discovery for infectious diseases of the developing worldKatsuno, 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.
- 34Chou, 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.58.3.10Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtVOhtLfL&md5=374c53e0772c679d1cdb08ff3112be90Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studiesChou, Ting-ChaoPharmacological 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.
- 35Jones, 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)80037-9Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXjtVKlsbc%253D&md5=bf45edafb986a73c7c1d0ac55e070f28Molecular recognition of a receptor sites using a genetic algorithm with a description of desolvationJones, 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.
- 36Jones, 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.1996.0897Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXis1KntLo%253D&md5=476a2b1d8f80f3ba418052fe29d735caDevelopment and validation of a genetic algorithm for flexible dockingJones, Gareth; Willett, Peter; Glen, Robert C.; Leach, Andrew R.; Taylor, RobinJournal 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.
- 37Schrödinger Release 2015-4: Maestro, version 10.4; Schrödinger, LLC: New York, 2015.Google ScholarThere is no corresponding record for this reference.
- 38Sanschagrin, 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.5560071002Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntVSlsrY%253D&md5=eb46b53aee05dfec17053634a0ce2980Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificitySanschagrin, 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.
- 39Small-Molecule Drug Discovery Suite 2015-4: Glide, version 6.9; Schrödinger, LLC: New York, 2015.Google ScholarThere is no corresponding record for this reference.
- 40Friesner, 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/jm051256oGoogle Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpvVGmurg%253D&md5=ea428c82ead0d8c27f8c1a7b694a1edfExtra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand ComplexesFriesner, 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.
- 41Halgren, 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/jm030644sGoogle Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyit78%253D&md5=33d68dd968e65626b449df61e44e37beGlide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screeningHalgren, 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.
- 42Friesner, 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/jm0306430Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhsFyit74%253D&md5=8cc2f0022318b12dd972e9c493375bf9Glide: A new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracyFriesner, 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.
- 43Li, 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.20660Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlaltLnN&md5=9e33529954c65867929326b99dac493bVery fast empirical prediction and rationalization of protein pKa valuesLi, 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.
- 44Cardinale, 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.1104829108Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFams7jM&md5=46b7c3dde0c96c0c3d053a58aa84856fProtein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthaseCardinale, 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. PaolaProceedings 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.
- 45Benvenuti, 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.2007.198Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtFahurzF&md5=609fceaec4d8b9b3ad5a129f57989e68Crystallization of soluble proteins in vapor diffusion for X-ray crystallographyBenvenuti, Manuela; Mangani, StefanoNature 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.
- 46Leslie, A. G. The integration of macromolecular diffraction data Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48– 57 DOI: 10.1107/S0907444905039107Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7vP&md5=70d65687f3b3426c93e96bfb706e0c5cThe integration of macromolecular diffraction dataLeslie, 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.
- 47Evans, P. Scaling and assessment of data quality Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 72– 82 DOI: 10.1107/S0907444905036693Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlant7jM&md5=293d3876e534c0c57813990515bb3c76Scaling and assessment of data qualityEvans, PhilipActa 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.
- 48Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760– 763. DOI: 10.1107/S0907444994003112Google ScholarThere is no corresponding record for this reference.
- 49Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement J. Appl. Crystallogr. 1997, 30, 1022– 1025 DOI: 10.1107/S0021889897006766Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhslehu7Y%253D&md5=96af64aedd8a8f018c3c18b57a6b4b21MOLREP: an automated program for molecular replacementVagin, Alexei; Teplyakov, AlexeiJournal 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.
- 50Dawson, 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/S0907444910040886Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFSgsr3O&md5=3d2805846022b2bf45531dd8f156786cHigh-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 targetDawson, 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.
- 51Murshudov, 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/S0907444996012255Google ScholarThere is no corresponding record for this reference.
- 52Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126– 2132 DOI: 10.1107/S0907444904019158Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVars73P&md5=1be390f3bb6fd584468499ad0921161eCoot: model-building tools for molecular graphicsEmsley, Paul; Cowtan, KevinActa 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'.
- 53Laskowski, 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/S0021889892009944Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXit12lurY%253D&md5=fb69fc4410cd716aaaa7cc0db06b3ed2PROCHECK: a program to check the stereochemical quality of protein structuresLaskowski, 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.
- 54McNicholas, 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/S0907444911007281Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqurw%253D&md5=d96403a39bc723a66f11a730f81f76c3Presenting your structures: The CCP4mg molecular-graphics softwareMcNicholas, 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.
- 55Shanks, 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.2009.09.003Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFShtb3N&md5=73a55624db0bf7bc04b768bef03807dfDevelopment and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductaseShanks, 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.
- 56Sereno, 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– 3102Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1M%252FlsVynug%253D%253D&md5=6e1275a26903f7ffdc91c68a458b8d0fAxenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of actionSereno D; Cavaleyra M; Zemzoumi K; Maquaire S; Ouaissi A; Lemesre J LAntimicrobial 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.
- 57Bowling, 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.2012.02.002Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cvls1egsg%253D%253D&md5=a8e04cd8cedfe8b8f505ff704ecce376Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasisBowling Tana; Mercer Luke; Jacobs Robert; Nare Bakela; Don RobertInternational 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.
- 58Bifeld, 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.Google ScholarThere is no corresponding record for this reference.
- 59Shia, 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/jf802378qGoogle Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsFWhsLzO&md5=111578f6be5b32f02343ccdc5a2509f2Metabolism and Pharmacokinetics of 3,3',4',7-Tetrahydroxyflavone (Fisetin), 5-Hydroxyflavone, and 7-Hydroxyflavone and Antihemolysis Effects of Fisetin and Its Serum MetabolitesShia, Chi-Sheng; Tsai, Shang-Yuan; Kuo, Sheng-Chu; Hou, Yu-Chi; Chao, Pei-Dawn LeeJournal 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.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe 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)
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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