SARS-CoV-2 Main Protease Drug Design, Assay Development, and Drug Resistance Studies.

SARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug-drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks.In this Account, we describe our efforts in developing covalent and noncovalent main protease (M pro ) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of M pro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing M pro -specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent M pro inhibitors such as Jun8-76-3R that are highly selective toward M pro over host cathepsin L. Using the same scaffold, we also designed covalent M pro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP M pro assay to characterize the cellular target engagement of our rationally designed M pro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate M pro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring M pro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent M pro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable M pro inhibitors that do not have overlapping resistance profile with nirmatrelvir.