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.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

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.