Insights into the Main Protease of SARS-CoV-2: Thermodynamic Analysis, Structural Characterization, and the Impact of Inhibitors

The global pandemic COVID-19, caused by the severe acute respiratory syndrome coronavirus (SARS- CoV-2), has taken a staggering toll on human health. The main protease of SARS-CoV-2 (M pro ) is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of M pro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193-nm ultraviolet photodissociation (UVPD) to structurally characterize M pro and its interactions with potential inhibitors. Melting temperatures and equilibrium constants, as well as the overall energy landscape, were obtained using variable temperature nano-electrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of M pro . The melting temperature was determined to be approximately 43°C for the dimer and 50°C for the monomer, suggesting an initial thermal dissociation pathway before subsequent unfolding of the monomer species. Thermodynamic parameters extracted from Van’t Hoff plots revealed dimeric complexes containing one of four inhibitors showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for potential inhibition mechanisms.