Rational Design of Macrocyclic Noncovalent Inhibitors of SARS-CoV-2 M from a DNA-Encoded Chemical Library Screening Hit That Demonstrate Potent Inhibition against Pan-Coronavirus Homologues and Nirmatrelvir-Resistant Variants.

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (M) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent M inhibitor was the first such approved therapy. Although M inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent M inhibitor , which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone with significantly improved antiviral activity. Further optimization led to the potent lactam , which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.