Allosteric inhibition of the SARS-CoV-2 main protease - insights from mass spectrometry-based assays.
Robinson CV., El-Baba TJ., Lutomski CA., Kantsadi AL., Malla TR., John T., Mikhailov V., Bolla JR., Schofield CJ., Zitzmann N., Vakonakis I.
Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease M pro cleaves at eleven sites to release non-structural proteins required for viral replication. M Pro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of M pro . Analysis of the monomer-dimer equilibria reveals a dissociation constant of K d = 0.14 ± 0.03 µM, revealing M Pro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively "flash-frozen" as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate M Pro activity.