Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening

BACKGROUND 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. OBJECTIVE 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. METHODS 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. FINDINGS 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). MAIN CONCLUSION Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Past and future of trypanosomatids high-throughput phenotypic screening

Diseases caused by trypanosomatid parasites affect millions of people mainly living in developing countries. Novel drugs are highly needed since there are no vaccines and available treatment has several limitations, such as resistance, low efficacy, and high toxicity. The drug discovery process is often analogous to finding a needle in the haystack. In the last decades a so-called rational drug design paradigm, heavily dependent on computational approaches, has promised to deliver new drugs in a more cost-effective way. Paradoxically however, the mainstay of these computational methods is data-driven, meaning they need activity data for new compounds to be generated and available in databases. Therefore, high-throughput screening (HTS) of compounds still is a much-needed exercise in drug discovery to fuel other rational approaches. In trypanosomatids, due to the scarcity of validated molecular targets and biological complexity of these parasites, phenotypic screening has become an essential tool for the discovery of new bioactive compounds. In this article we discuss the perspectives of phenotypic HTS for trypanosomatid drug discovery with emphasis on the role of image-based, high-content methods. We also propose an ideal cascade of assays for the identification of new drug candidates for clinical development using leishmaniasis as an example.