The Function and Inhibition of SARS-CoV-2 Main Protease (Mpro)

Abstract

The SARS-CoV-2 main protease (Mpro) is essential for viral replication and has no close human counterpart, making it a prime antiviral target. Yet the chemical space of covalent Mpro inhibitors remains narrow, and how these molecules recognize the active site, react with the catalytic cysteine, and are affected by the enzyme's other reactive residues is incompletely understood. This thesis addresses these questions across inhibitor discovery, enzyme engineering, and computational design. Cyclobutanone, a ring-strained electrophile inspired by penicillin's mechanism, is first shown to be a viable non-peptidic warhead for Mpro: a synthesized series inhibits the enzyme in a time-dependent manner consistent with covalent engagement, with potency governed by substituent occupancy of the hydrophobic S2–S4 subsites rather than by ring saturation. To resolve whether inhibitor potency is masked by off-target reactivity, an Mpro variant lacking its three solvent-exposed, non-catalytic cysteines is engineered. This mutant retains the wild-type fold and active-site geometry by crystallography, yet its apparent sensitivity to a covalent reference inhibitor change markedly, revealing that surface cysteines act as competing decoys in the native enzyme. The catalytic dyad itself is then dissected, with mutagenesis and crystallography showing that its two residues are functionally distinguishable: disabling the general base leaves substrate-groove geometry and covalent inhibitor trapping intact, whereas disabling the nucleophile is accompanied by localized structural rearrangement, indicating asymmetric roles in inhibitor recognition. Finally, applying molecular docking, dynamics, and free-energy calculations to a new aldehyde-based series shows that docking score and single-point binding energy are poor predictors of true potency: two computationally top-ranked compounds are among the weakest experimentally, an inconsistency resolved only by tracking pose stability across simulated time, which reveals their failure to remain productively bound. Together, these findings identify two new covalent Mpro inhibitor chemotypes, provide validated tools for isolating genuine catalytic-site reactivity, and establish binding-pose persistence, not static computational scores, as essential for reliable inhibitor triage.