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In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors.

ABSTRACT: Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CL^pro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CL^pro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CL^pro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme's putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer.

SUBMITTER: Guo J

PROVIDER: S-EPMC7365090 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSchistosomiasis 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.ObjectiveIn 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.MethodsInitially, 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.FindingsSeven 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 conclusionOverall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

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In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors.

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