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 (M ^pro ) is a key target for antiviral drugs. Given its conserved sequence across coronaviruses and its essential role in viral replication, numerous inhibitors have been developed to target its active site. M ^pro exists in equilibrium between the active dimer and inactive monomer, rendering the targeting of dimerization as a promising alternative strategy for drug development. This study addresses knowledge gaps regarding the monomer-dimer equilibrium and conformational changes of M ^pro induced by inhibitor binding. We utilized ¹³ C labeling combined with native mass spectrometry to assess how different types of inhibitors (including peptidomimetic inhibitors PF-07321332, PF-00835231, GC376, boceprevir; non-peptidomimetic inhibitors carmofur, ebselen and its analog MR6-31-2; and allosteric inhibitors AT7519 and pelitinib) influence the monomer-dimer equilibrium and subunit exchange of M ^pro . Additionally, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to investigate the conformational dynamics of M ^pro 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 M ^pro dimer interface. 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 M ^pro inhibitors.