Dynamical Nonequilibrium Molecular Dynamics Simulations Identify Allosteric Sites and Positions Associated with Drug Resistance in the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (M pro ) plays an essential role in the coronavirus lifecycle by catalyzing hydrolysis of the viral polyproteins at specific sites. M pro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how M pro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of M pro to the presence and absence of a substrate. The results highlight communication between the M pro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behavior of M pro . More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.