Molecular Basis for Antiviral Action of EDP-235: A Potent and Selective SARS-CoV-2 3clpro Inhibitor for the Treatment of Covid 19.

To date, there are no approved oral antiviral therapies that can be administered early in the course of COVID-19 to suppress progression of the disease or for prophylaxis. EDP-235 is a potent and selective inhibitor of SARS-CoV-2 3C-like protease (3CLpro). EDP-235 inhibits SARS-CoV-2 3CLpro protease activity with an IC of 5.8 ± 3.7 nM and retains its activity against variant 3CLpro proteins from multiple SARS-CoV-2 lineages (IC range of 2.8--5.8 nM). 3CLpro protease activity progress curves showed significant curvature in a time- and EDP-235-concentration-dependent manner indicative of slow-onset inhibition. Slow reversal of inhibition of SARS-CoV-2 3CLpro enzyme activity was observed in a jump dilution experiment. Michaelis-Menten kinetic studies with a FRET peptide substrate in the presence of EDP-235 indicated that EDP-235 is a substrate-competitive inhibitor of SARS-CoV-2 3CLpro with an overall dissociation constant K of 3.0 ± 1.6 nM. SARS-CoV-2 3CLpro was crystallized bound to a close analog of EDP-235 and structure elucidation revealed that the ligand bound at the active site and interacted with side chains of conserved residues Cys-145, His-163, and Glu-166. EDP-235 also potently inhibits 3CLpro enzymes from other α-coronaviruses (IC range of 2-4 nM) and β-coronaviruses (SARS-CoV IC of 5.4 nM, MERS-CoV IC of 70 nM) which cause disease in humans to date. EDP-235 resistance mutations in HCoV-229E map to the active site of 3CLpro close to the predicted binding site and offer additional support to the mechanism of inhibition. EDP-235 also showed a favorable selectivity profile (>300 selectivity index) when tested against a panel of 30 mammalian proteases. In summary, EDP-235 acts as a slow-onset, slow-reversible, substrate-competitive inhibitor of SARS-CoV-2 3CLpro. The outstanding preclinical profile of EDP-235 supports its further evaluation as an oral therapeutic for the management of COVID-19.