PROTAC-based approach to develop broad-spectrum antiviral agents triggering the proteolysis of the major viral protease

In 2020, with the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, PROteolysis TArgeting Chimera (PROTAC) degraders acting against the major viral protease 3-chymotrypsin-like protease (3CLPro) were hypothesized to be next-generation anti-coronavirus drugs. 3CLPro is shared by all coronavirus genera as well as by members of the large genus Enterovirus in the Picornavirus family. Combining NMR spectroscopy and X-ray crystallography, we characterize the interaction between the α, β-unsaturated peptidomimetic-based PROTAC (FT235) with 3CLPro from SARS-CoV-2 and 3CPro from Coxsackievirus B3 (CVB3), the latter being a cardiotropic virus belonging to enterovirus genus. The results are compared with those obtained with the precursor molecule (FT234). We demonstrate that PROTAC molecule binds to the active site of both enzymes and the residues involved in the interaction are identified by solution NMR experiments. Furthermore, the crystal structures of the main proteases from SARS-CoV-2 and CVB3 in complex with both FT234 and FT235 molecules are obtained. For both ligands the Cβ carbon of the α, β-unsaturated amide moiety forms a covalent bond with the catalytic cysteines. Finally, preliminary cellular studies show that the synthetized PROTAC molecule drastically reduces protein levels of SARS-CoV-2 3CLPro in cultured cells.