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کشف یک داروی ضدانگلی جدید

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انگل‌های تریپانوزوماتید موجب بیماری‌هایی مانند بیماری خواب آفریقایی، بیماری شاگاس و لیشمانیازیس می‌شوند. لیشمانیازیس ۱۲ میلیون نفر را در دنیا مبتلا می‌کند که عمدتاً در کشور‌های در حال توسعه هستند. درمان‌های دارویی کنونی مورد استفاده برای این بیماری‌ها، به دلیل سمیّت زیاد و مقاومت دارویی، نامناسب هستند.
اکنون یک گروه از دانشمندان اروپایی ترکیبات جدیدی را کشف کرده‌اند که می تواند به طرز مؤثری به جنگ بر علیه این بیماری‌ها کمک کند. نتایج این پروژه در مجله شیمی پزشکی (Journal of Medicinal Chemistry) منتشر شده است.
تریپانوزوماتیدها به فولات (folates) و بیوپترین‌ها (biopterins) نیاز دارند. این مواد به وسیله آنزیم‌های دی‌هیدروفولات ردوکتاز (DHFR) و پتریدین ردوکتاز (PTR1) احیاء می‌شوند. اگر DHFR مهار شود، تکثیر DNA آسیب می‌بیند که به مرگ سلول می‌انجامد. اما در تریپانوزوماتیدها در صورت مهار DHFR، میزان بروز PTR1 افزایش یافته و کمبود آنزیم DHFR را جبران کرده و از طریق احیای فولات، بقای انگل را تضمین می کند. بنابراین برای این بیماری‌های انگلی لازم است که هر دو مسیر آنزیمی فوق به صورت هم‌زمان، با یک داروی واحد یا ترکیبی از دو مهارکننده اختصاصی مهار شوند. در انسان‌ها، PTR1  وجود ندارد و لذا یک هدف عالی برای طراحی داروهای اختصاصی ضد انگلی است.
در این پروژه، دانشمندان پس از بهینه‌سازی روش‌ها و بررسی‌های دقیق روی ۱۸ ملکول شبه دارو، نهایتاً به ترکیبی رسیدند که یک داروی شناخته شده برای درمان بیماری‌های سیستم عصبی مرکزی است که به نظر می‌رسد با تغییراتی جزئی در آن، می‌تواند کاندیدای مناسبی به عنوان یک داروی انگلی باشد.

Abstract

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules 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 compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 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 preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.

Introduction


Parasites of the Trypanosomatidae family are the causal agents of a number of serious human diseases, including African sleeping sickness, Chagas’ disease, and leishmaniasis. The impact of these parasites on public health and the inadequacy of current treatments have created an urgent requirement for more effective drugs, since those in use are highly toxic and often difficult to administer. The problem is compounded by a rise in drug resistance. Therapy for leishmaniasis generally relies on old drugs such as sodium stibogluconate and Amphotericin B, and only one new treatment has been developed in the last 25 years, Miltefosine, which is approved in India for visceral Leishmanisis.(1)
Enzymes involved in the provision of reduced folate cofactors, e.g., dihydrofolate reductase (DHFRa
a

Abbreviations: ACD, Available Chemicals Directory; DHFR, dihydrofolate reductase; PTR1, pteridine reductase; rmsd, root-mean-square deviation; TS, thymidylate synthase.

), and enzymes that utilize these cofactors, like thymidylate synthase (TS), are important drug targets for the treatment of bacterial infections,(2) cancer,(3) and certain parasitic diseases, notably malaria.(4) Inhibition of DHFR or TS leads to a reduction in the cellular pools of 2′-deoxythymidine-5′-monophosphate, severely impairs DNA replication, and results in cell death. Trypanosomatids are auxotrophic for folates and pterins, and inhibition of the enzymes involved in the salvage pathways should provide effective treatment.(5) However, antifolates are currently not employed in therapy of trypanosomatid infections, mainly because of the pteridine reductase (PTR1) activity of the target organisms. PTR1 is a short-chain dehydrogenase/reductase that is able to carry out successive reductions of both conjugated (folate) and unconjugated (biopterin) pterins.(6, 7) While the bifunctional DHFR-TS used by trypanosomatids can only reduce folic acid, PTR1 can act on a broader range of substrates. Under physiological conditions, PTR1 is responsible for the reduction of 10% of the folic acid required by the cell, but when classical antifolate drugs inhibit DHFR-TS, PTR1 can be overexpressed, ensuring parasite survival.(8, 9) This suggests that treatment of trypanosomatid infections could be achieved through the simultaneous inhibition of DHFR and PTR1 by a single drug or a combination of compounds that are specific and selective inhibitors of both targets.(8)
Previous work in our laboratories(10) has demonstrated that it is possible to identify specific inhibitors of PTR1 and to use such inhibitors in combination with known antifolates to effectively improve in vitro efficacy against Leishmania and Trypanosoma species.(10) A similar approach has been taken to discover new compounds with pyrrolo[2,3-d]pyrimidine structures that are active against Trypanosoma brucei PTR1 and on bloodstream parasites.(11) Very recently, a virtual screening strategy revealed compounds with aminobenzothiazole and aminobenzimidazole scaffolds that inhibit Trypanosoma brucei PTR1.(12) In the present paper, we describe the application of a different virtual screening approach combined with rapid synthetic and experimental screening methodologies that has enabled us to identify nonfolate like inhibitors of Leishmania PTR1 with thiadiazole core structures. The hits identified have been optimized in two structure-based design cycles, including species specificity studies. The compounds were tested against the Leishmania major enzymes, LmPTR1 and LmDHFR-TS, and the human DHFR (hDHFR). The best compounds were also assayed against in vitro cultured cells of different trypanosomatid species, such as promastigotes of L. mexicana and L. major, both as single agents and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine)(13) (Supporting Information Figure 1-SI). The toxicity of these compounds was tested against human fibroblasts (MRC5).

Results and Discussion


Virtual Screening Hits and Scaffold Identification
Virtual screening of the Available Chemicals Directory (ACD) database against the three-dimensional structure of LmPTR1 (PDB ID: 1E92) using the program LUDI(14) resulted in 21394 docked molecules. Analysis of the available crystallographic structures showed that the substrate, dihydrobiopterin binds to LmPTR1 by forming an extended network of hydrogen bonds and aromatic stacking interactions with the cofactor and Phe113. During the selection of the docked molecules, we wanted to select compounds able to mimic the binding mode of this substrate. The docked molecules were initially filtered based on three calculated parameters: (I) the ability of the ligand to bind deep into the active site (contact percentage >50); (II) the ability to replace the substrate by forming hydrogen bonds with the active site residues (number of H bonds >1); (III) the calculated score (>400). The remaining 724 docking results were analyzed visually and molecules were selected based on (I) the number and type of interactions established, (II) which residues they interact with, and (III) comparison of the structures of the active sites of LmPTR1 and hDHFR. Molecules that were predicted to form a stacking interaction with Phe113 and to interact with LmPTR1 residues that are not conserved in the hDHFR active site were selected preferentially. This second selection resulted in 53 molecules that were purchased and tested against LmPTR1 (Supporting Information Table 1-SI). Six of these compounds (4a, 6a, 28a, 35a, 38a, 53a) were found to be active against LmPTR1 with IC50 values between 0.39 and 5.6 mM (Table 1, Figure 1). These molecules were further tested for their inhibitory activity against LmDHFR and hDHFR. None of them was active against hDHFR. Only compound 28a showed weak inhibitory activity against LmDHFR. Compound 4a was selected for further development on the basis of synthetic feasibility and biological activity (IC50 and Ki against LmPTR1 of 5.6 mM and 436 μM, respectively). The derivatization of the hit at position 5 of the thiadiazole ring (Figure 2) was expected to increase the affinity and the specificity for PTR1 by introduction of fragments that lead to more complex and drug-like compounds
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