Distinct Mechanisms for Inhibition of SARS-CoV-2 Main Protease: Dimerization Promoted by Peptidomimetic Inhibitors and Disrupted by Ebselen

The SARS-CoV-2 main protease (Mpro) is a key target for antiviral drugs. Given its conserved sequence across coronaviruses and essential role in viral replication, numerous inhibitors have been developed to target its active site. Mpro exists in equilibrium between the active dimer and inactive monomer, rendering targeting dimerization as a promising alternative strategy for drug development. This study investigates the monomer-dimer equilibrium and conformational changes of Mpro induced by inhibitor binding. We utilized 13C labeling combined with native mass spectrometry to assess how different inhibitors, including peptidomimetic inhibitors (PF-07321332, PF-00835231, GC376, and boceprevir), and non-peptidomimetic inhibitors (carmofur, ebselen, MR6-31-2, AT7519 and pelitinib), influence Mpro dimerization and subunit exchange. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was further employed to investigate the conformational dynamics of Mpro and its interactions with these inhibitors. Key findings revealed divergent mechanisms: peptidomimetic inhibitors significantly shifted the equilibrium towards the dimeric state, suppressing subunit exchange dynamics and rigidifying the dimer interface. In contrast, ebselen impaired the dimer form and increased the flexibility of the dimer interface. Notably, we identified a novel covalent binding site for ebselen at C300 by tandem mass spectrometry, with molecular dynamics simulations further indicating that this modification allosterically altered the hydrogen bond network of the Mpro dimer interface. Importantly, C300 mutational analyses demonstrated that C300 contributes to ebselen-mediated dimer disruption and enzymatic inhibition. Overall, this study reveals distinct inhibitory modes between peptidomimetic inhibitors and ebselen, highlighting the potential of targeting allosteric sites at the dimer interface for the design of next-generation Mpro inhibitors.