Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2

The 3C-like protease (3CL^pro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CL^pro as non-peptidomimetic covalent binders at submicromolar levels, with IC₅₀ values ranging from 0.118 to 0.582 μM. Interestingly, these compounds were also shown to inhibit PL^pro with the same level of IC₅₀ values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC₅₀ value of 7.249 μM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CL^pro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.