Abstract:
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 (Mpro) to adapt and develop resistance against nirmatrelvir.
Employing a unique high-throughput protein design technique, the hotspot residues, and signatures of
adaptation of Mpro having the highest probability of mutating and rendering nirmatrelvir ineffective
were identified. Our results show that ~40% of the designed mutations in Mpro already exist in the
globally circulating SARS-CoV-2 lineages and several predicted mutations. Moreover, several highfrequency, 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 Mpro and is thus crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving Mpro variations.
