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

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 UAWJ9-36-1 and UAWJ9-36-3 based on the superimposed X-ray crystal structures of SARS-CoV-2 M-Pro with GC-376, telaprevir, and boceprevir. Both UAWJ9-36-1 and UAWJ9-36-3 showed potent binding and enzymatic inhibition against the M-Pro 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 UAWJ9- 36-1 and UAWJ9-36-3 inhibited the intracellular protease activity of SARS-CoV-2 M-Pro. In addition, UAWJ9-36-1 and UAWJ9-363 had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with UAWJ9-36-3 being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that UAWJ9-36-1 and UAWJ9-36-3 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 UAWJ9-36-1 and UAWJP-36-3 were both solved at 1.9 angstrom, which validated our design hypothesis. Overall, hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 are promising candidates to be further developed as broad-spectrum coronavirus antivirals.