Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease

Nirmatrelviris an orally available inhibitor of SARS-CoV-2mainprotease (Mpro) and the main ingredient of Paxlovid, a drug approvedby the U.S. Food and Drug Administration for high-risk COVID-19 patients.Recently, a rare natural mutation, H172Y, was found to significantlyreduce nirmatrelvir's inhibitory activity. As the COVID-19cases skyrocket in China and the selective pressure of antiviral therapybuilds in the US, there is an urgent need to characterize and understandhow the H172Y mutation confers drug resistance. Here, we investigatedthe H172Y Mpro's conformational dynamics, folding stability,catalytic efficiency, and inhibitory activity using all-atom constantpH and fixed-charge molecular dynamics simulations, alchemical andempirical free energy calculations, artificial neural networks, andbiochemical experiments. Our data suggest that the mutation significantlyweakens the S1 pocket interactions with the N-terminus and perturbsthe conformation of the oxyanion loop, leading to a decrease in thethermal stability and catalytic efficiency. Importantly, the perturbedS1 pocket dynamics weaken the nirmatrelvir binding in the P1 position,which explains the decreased inhibitory activity of nirmatrelvir.Our work demonstrates the predictive power of the combined simulationand artificial intelligence approaches, and together with biochemicalexperiments, they can be used to actively surveil continually emergingmutations of SARS-CoV-2 Mpro and assist the optimization of antiviraldrugs. The presented approach, in general, can be applied to characterizemutation effects on any protein drug targets.