Structure-Guided Discovery and Biochemical Validation of Novel Small-Molecule Inhibitors Predicted to Target the CCHFV OTU Protease Y89-W99 Pocket

Crimean–Congo hemorrhagic fever virus (CCHFV) remains a major public health threat due to its high mortality rates and the absence of approved antiviral therapies. The viral ovarian tumor (OTU) protease is a critical virulence factor that suppresses host innate immunity through its deubiquitinase activity, making it an attractive therapeutic target. In this study, we employed a structure-guided approach to identify and validate novel small-molecule inhibitors targeting the non-catalytic Y89-W99 pocket of the OTU protease. Recombinant OTU protease was successfully expressed, purified, and refolded, yielding a soluble and enzymatically active protein. Cellular assays confirmed that the enzyme retains robust deubiquitinase activity, significantly reducing global ubiquitin conjugates in mammalian cells. In silico analysis of a putative DUB inhibitor library identified several candidate inhibitors with favorable binding interactions within the Y89-W99 pocket. Biochemical validation using a fluorometric Ub-AMC assay revealed that multiple small molecules strongly inhibit OTU activity, including OTUi-10 (~93% inhibition), OTUi-13 (~87%), OTUi-1 (~85%), OTUi-4 and OTUi-11 (~81%), and OTUi-9 (~76%). Additional moderate inhibitors included OTUi-12 (~67%), OTUi-19 and OTUi-21 (~66%), and OTUi-5 (~57%). In silico drug-likeness and toxicity profiling filtered the library to four fully compliant candidates, OTUi-4, OTUi-10, OTUi-11, and OTUi-12, all free of predicted toxicity alerts. These findings suggest that the Y89–W99 pocket may be a pharmacologically relevant site worthy of further investigation and identify OTUi-10, OTUi-4, and OTUi-11 as promising preliminary hit compounds. The results also provide initial insights that may guide future optimization and mechanistic studies of OTU protease inhibitors targeting CCHFV.