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

The main protease (Mpro) of SARS-CoV-2 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 Mpro as viral resistance
to Paxlovid emerges. Here, we report the use of native mass spectrometry
coupled with 193 nm ultraviolet photodissociation (UVPD) and integrated
with other biophysical tools to structurally characterize Mpro and its interactions with potential covalent inhibitors. The overall
energy landscape was obtained using variable temperature nanoelectrospray
ionization (vT-nESI), thus providing quantitative evaluation of inhibitor
binding on the stability of Mpro. Thermodynamic parameters
extracted from van’t Hoff plots revealed that the dimeric complexes
containing each inhibitor showed enhanced stability through increased
melting temperatures as well as overall lower average charge states,
giving insight into the basis for inhibition mechanisms.