Rational Design of Hybrid SARS-CoV-2 Main Protease Inhibitors Guided by the Superimposed Cocrystal Structures with the Peptidomimetic Inhibitors GC-376, Telaprevir, and Boceprevir

Abstract

SARS-CoV-2 main protease (M^pro) is a cysteine protease that mediates the cleavage of viral polyproteins and is a validated antiviral drug target. M^pro is highly conserved among all seven human coronaviruses, with certain M^pro inhibitors having broad-spectrum antiviral activity. In this study, we designed two hybrid inhibitors <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> based on the superimposed X-ray crystal structures of SARS-CoV-2 M^pro with GC-376, telaprevir, and boceprevir. Both <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> showed potent binding and enzymatic inhibition against the M^pro's from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. Cell-based Flip-GFP M^pro assay results show that <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> inhibited the intracellular protease activity of SARS-CoV-2 M^pro. In addition, <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with <b>UAWJ9-36-3</b> being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> had an improved selectivity index over that of GC-376 against host cysteine proteases calpain I and cathepsin L, but not cathepsin K. The X-ray crystal structures of SARS-CoV-2 M^pro with <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> were both solved at 1.9 Å, which validated our design hypothesis. Overall, hybrid inhibitors <b>UAWJ9-36-1</b> and <b>UAWJ9-36-3</b> are promising candidates to be further developed as broad-spectrum coronavirus antivirals.