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Nirmatrelvir, a pivotal component of the oral antiviral Paxlovid for COVID-19, targets the SARS-CoV-2 main protease (M pro ) as a covalent inhibitor. Here, we employed combined computational methods to explore how the prevalent Omicron variant mutation P132H, alone and in combination with A173V (P132H-A173V), affects nirmatrelvir's efficacy. Our findings suggest that P132H enhances the noncovalent binding affinity of M pro for nirmatrelvir, whereas P132H-A173V diminishes it. Although both mutants catalyze the rate-limiting step more efficiently than the wild-type (WT) M pro , P132H slows the overall rate of covalent bond formation, whereas P132H-A173V accelerates it. Comprehensive analysis of noncovalent and covalent contributions to the overall binding free energy of the covalent complex suggests that P132H likely enhances M pro sensitivity to nirmatrelvir, while P132H-A173V may confer resistance. Per-residue decompositions of the binding and activation free energies pinpoint key residues that significantly affect the binding affinity and reaction rates, revealing how the mutations modulate these effects. The mutation-induced conformational perturbations alter drug-protein local contact intensities and the electrostatic preorganization of the protein, affecting noncovalent binding affinity and the stability of key reaction states, respectively. Our findings inform the mechanisms of nirmatrelvir resistance and sensitivity, facilitating improved drug design and the detection of resistant strains.

Psychrophilic enzymes were always observed to have higher catalytic activity ( k cat ) than their mesophilic homologs at room temperature, while the origin of this phenomenon remains obscure. Here, we used two different temperature-adapted trypsins, the psychrophilic Atlantic cod trypsin (ACT) and the mesophilic bovine trypsin (BT), as a model system to explore the energetic origin of their different catalytic activities using computational methods. The results reproduce the characteristic changing trends in the activation free energy, activation enthalpy, and activation entropy between the psychrophilic and mesophilic enzymes, where, in particular, the slightly decreased activation free energy of ACT is determined by its considerably reduced activation enthalpy rather than by its more negative activation entropy compared to BT. The calculated electrostatic contributions to the solvation free energies in the reactant state/ground sate (RS/GS) and transition state (TS) show that, going from BT to ACT, the TS stabilization has a predominant effect over the RS stabilization on lowering the activation enthalpy of ACT. Comparison between the solvation energy components reveals a more optimized electrostatic preorganization to the TS in ACT, which provides a larger stabilization to the TS through reducing the reorganization energy, thus resulting in the lower activation enthalpy and hence lower activation free energy of ACT. Thus, it can be concluded that it is the difference in the protein electrostatic environment, and hence its different stabilizing effects on the TS, that brings about the different catalytic activities of different temperature-adapted trypsins., Competing Interests: The authors declare no competing financial interest.