Rational Design\nof Macrocyclic Noncovalent Inhibitors\nof SARS-CoV‑2 M<sup>pro</sup> from a DNA-Encoded Chemical Library\nScreening Hit That Demonstrate Potent Inhibition against Pan-Coronavirus\nHomologues and Nirmatrelvir-Resistant Var

The recent global COVID-19 pandemic\nhas highlighted treatments\nfor coronavirus infection as an unmet medical need. The main protease\n(M<sup>pro</sup>) has been an important target for the development\nof SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent\nM<sup>pro</sup> inhibitor was the first such approved therapy. Although\nM<sup>pro</sup> inhibitors of various chemical classes have been reported,\nthey are generally less active against nirmatrelvir-resistant variants\nand have limited pan-coronavirus potential, presenting a significant\nhuman health risk upon future outbreaks. We here present a novel approach\nand utilized DNA-encoded chemical library screening to identify the\nnoncovalent M<sup>pro</sup> inhibitor <b>5</b>, which demonstrated\na distinct binding mode to nirmatrelvir. A macrocyclization strategy\ndesigned to lock the active conformation resulted in lactone <b>12</b> with significantly improved antiviral activity. Further\noptimization led to the potent lactam <b>26</b>, which demonstrated\nexceptional potency against nirmatrelvir-resistant variants as well\nas against a panel of viral main proteases from other coronaviruses.