Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes

Shortly after the onset of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired numerous variations in its intracellular proteins to adapt quickly, become more infectious, and ultimately develop drug resistance by mutating certain hotspot residues. To keep the emerging variants at bay, including Omicron and subvariants, FDA has approved the antiviral nirmatrelvir for mild-to-moderate and high-risk COVID-19 cases. Like other viruses, SARS-CoV-2 could acquire mutations in its main protease (M(pro)) to adapt and develop resistance against nirmatrelvir. Employing a unique high-throughput protein design technique, the hotspot residues, and signatures of adaptation of M(pro) having the highest probability of mutating and rendering nirmatrelvir ineffective were identified. Our results show that ∼40% of the designed mutations in M(pro) already exist in the globally circulating SARS-CoV-2 lineages and several predicted mutations. Moreover, several high-frequency, designed mutations were found to be in corroboration with the experimentally reported nirmatrelvir-resistant mutants and are naturally occurring. Our work on the targeted design of the nirmatrelvir-binding site offers a comprehensive picture of potential hotspot sites and resistance mutations in M(pro) and is thus crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving M(pro) variations.