OTUD1 x OTUD7B x UCHL5 x USP7

Crimean-Congo hemorrhagic fever (CCHF) is a viral tick-borne disease with fatality rates of up to 30 %. Currently, there are no vaccines or specific antivirals available. The genome of the CCHF virus (CCHFV) encodes an ovarian tumor (OTU) protease with a deubiquitinating activity that is responsible for the evasion of the innate immune response. Therefore, the inhibition of the OTU protease could provide a strategy for the treatment of CCHFV infections. In this study, we screened for small-molecule inhibitors of CCHFV OTU using a fluorescent ubiquitin rhodamine 110 assay. We identified and validated a 2-aminothiazole hit compound (IC₅₀ = 42.3 μM) followed by structure-activity relationships (SAR) studies resulting in a new inhibitor of the CCHFV OTU protease. The most active derivative is a competitive CCHFV OTU inhibitor with an IC₅₀ value of 10.7 μM. Selectivity studies revealed that the ubiquitin-specific peptidase 7 (USP7), ubiquitin C-terminal hydrolase 5 (UCHL5), OTU deubiquitinase 1 (OTUD1), and Cezanne are also inhibited by this newly developed inhibitor indicating binding to conserved regions of the ubiquitin-binding site within the deubiquitinase superfamilies. Molecular docking into the active site of CCHFV OTU proposes starting points for further structural modifications to improve activity and selectivity. These structure-activity relationships are the first to our knowledge to be reported for the CCHFV OTU protease and will help guide further drug discovery efforts.