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) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of M 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-specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent M inhibitors such as Jun8-76-3R that are highly selective toward M over host cathepsin L. Using the same scaffold, we also designed covalent M 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 assay to characterize the cellular target engagement of our rationally designed M inhibitors. The FlipGFP assay was also applied to validate the structurally disparate M inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring M mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent M inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable M inhibitors that do not have overlapping resistance profile with nirmatrelvir.