Inhibition of Subunit Exchange and N-Finger Rearrangement in SARS-CoV-2 Main Protease by Peptidomimetic Inhibitors

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for viral replication, making it a critical target for antiviral drug development. While current Mpro inhibitors, such as Paxlovid, focus on active site binding, resistance mutations are emerging, underscoring the need for alternative strategies. Dimerization of Mpro, necessary for its enzymatic activity, presents a promising but underexplored target for drug development. However, knowledge gaps remain in understanding the dynamic conformational changes and network of residues that stabilize the dimer interface. In this study, we captured the transient dynamics of Mpro dimerization and the conformational changes at the dimer interface induced by inhibitor binding. Integrat-ing stable isotope labeling and native mass spectrometry with hydrogen-deuterium exchange, we provided time-resolved insights into the structural dynamics of Mpro in its intact dimeric form. We employed N15 isotopic labeling of Mpro to investi-gate subunit exchange and dissociation kinetics during dimerization, both in the presence and absence of peptidomimetic inhibitors, including Nirmatrelvir, GC376, and MPI8. Our findings revealed that peptidomimetic inhibitor binding inhibited dimer dissociation, altering the dynamics of subunit exchange. Moreover, we identified significant conformational changes across Mpro's three domains and at the dimer interface upon inhibitor binding. Notably, hydrogen-deuterium exchange pro-files transitioned from bimodal to unimodal distributions in the N-finger region when Mpro was complexed with Nirmatrelvir, suggesting distinct structural alterations that stabilize the dimeric form. By filling critical knowledge gaps in the understand-ing of Mpro dimerization and its inhibitor-induced dynamics, our study provides a foundation for the rational design of next-generation Mpro inhibitors targeting the dimer interface.