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

Abstract

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 IC50 of 5.8 ± 3.7 nM and retains its activity against variant 3CLpro proteins from multiple SARS‐CoV‐2 lineages (IC50 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 Ki 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 (IC50 range of 2–4 nM) and β‐coronaviruses (SARS‐CoV IC50 of 5.4 nM, MERS‐CoV IC50 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.