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

Since the onset of the COVID-19 pandemic, SARS-CoV-2 has acquired numerous variations in its intracellular proteins to quickly adapt, 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. The work provides a first-hand explanation of the resistance mutations in Mpro and is crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving Mpro variations.