Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Bioquímica Experimental e Computacional de Fármacos, Rio de Janeiro, RJ, Brasil.
Universidade Federal de Goiás, Faculdade de Farmácia, Laboratório de Planejamento de Fármacos e Modelagem Molecular, Goiânia, GO, Brasil.
Mem Inst Oswaldo Cruz. 2023 Sep 1;118:e230031. doi: 10.1590/0074-02760230031. eCollection 2023.
Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms.
In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors.
Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays.
Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 μM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 μM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h).
Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.
血吸虫病是一种由血吸虫属吸虫引起的被忽视的热带病，治疗方法有限，主要基于使用吡喹酮（PZQ）。目前，血吸虫种的基因组中已经鉴定出几种天冬氨酸蛋白酶基因。至少有一种来自这个基因家族的酶（SmAP），名为 SmCD1，已被验证可用于开发杀血吸虫药物，因为它在蠕虫消化血红蛋白中起着关键作用。
在这项工作中，我们整合了基于结构的虚拟筛选活动、酶测定和成虫体外实验，旨在发现 SmCD1 的第一类抑制剂。
最初，使用同源建模方法生成了 SmCD1、SmCD2 和 SmCD3 的 3D 结构。使用这些模型，我们从 ChemBridge 数据库的 20,000 种化合物中优先选择了 50 种化合物，用于进一步在成虫水提取物（AWAE）和重组 SmCD1 中进行酶测定。
有 7 种化合物被确认为命中化合物，其中两种代表新的化学骨架的化合物，命名为 5 和 19，对 SmCD1 的 IC50 值接近 100 μM，同时具有与胃蛋白酶抑制剂相当或甚至更高的结合效率指数。在与哺乳动物酶的活性比较中，化合物 50 是选择性的，对 AWAE 天冬氨酸蛋白酶活性的抑制作用最强（IC50 = 77.7 μM）。计算和实验结果的组合表明，化合物 50 是 SmCD2 的选择性抑制剂。在低浓度（10 μM）下测试的化合物 5、19 和 50 对 WSS-1 细胞（48 h）既没有细胞毒性，也不能在体外杀死成虫，尽管化合物 5 和 50 在后期孵育时间（48 或 72 h）对雌虫的运动有轻微的抑制作用。
总的来说，本研究中鉴定的抑制剂代表了进一步进行从头优化的有希望的命中化合物。
