The Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19

Abstract

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

“…Since the active sites of 3CLpro are fairly well conserved across different coronaviruses, it was hypothesized that PF-00835231 could be active against other coronaviruses besides SARS-CoV (15,18). Consistent with this hypothesis, it was shown that PF-00835231 was also active against 3CLpros from SARS-CoV-2 and HCoV-229E (15).…”

“…PF-00835231 exhibits tight and specific binding to SARS-CoV-2 3CL in vitro Previously, it was shown that PF-00835231 potently inhibited SARS-CoV-2 3CLpro with a Ki value of 0.27±0.1nM (15). An X-ray co-crystal structure of PF-00835231 bound to SARS-CoV-2 3CLpro is consistent with PF-00835231 binding to the 3CLpro enzyme with a covalent and reversible interaction at the catalytic cysteine residue of the active site, thus inhibiting the activity of the 3CLpro (PDB-6XHM) (15). A thermal-shift assay was used to evaluate the direct binding between PF-00835231 and SARS-CoV-2 3CLpro.…”

“…Since the active sites of 3CLpro are fairly well conserved across different coronaviruses, it was hypothesized that PF-00835231 could be active against other coronaviruses besides SARS-CoV (15,18). Consistent with this hypothesis, it was shown that PF-00835231 was also active against 3CLpros from SARS-CoV-2 and HCoV-229E (15). To further explore the notion that PF-00835231 could have pan-coronavirus activity, PF-00835231 was evaluated against 3CLpro from a variety of other coronaviruses representing alpha, beta and gamma groups of Coronaviridae, using biochemical Förster Resonance Energy Transfer (FRET) protease activity assays.…”

“…2). However, Vero cells express high levels of the efflux transporter P-glycoprotein (P-gp) (also known as MDR1 or ABCB1) (20), of which PF-00835231 is a known substrate (15) suggesting that the intracellular concentration of PF-00835231 was lower than the concentration added. Therefore, the assays were repeated in the presence of a P-gp efflux inhibitor, CP-100356 (21).…”

“…Following the severe acute respiratory syndrome (SARS) outbreak in 2002-2003 we identified a potential small molecule protease inhibitor (PF-00835231) for the treatment of SARS-CoV, using structure based drug design (15). Due to the SARS pandemic being brought under control in July 2003 following public health measures which incorporated patient isolation and travel restrictions, this project was discontinued.…”