Abstract
The SARS-CoV-2 main protease (M(pro) ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. M(Pro) is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry 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, indicating M(Pro) has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate M(Pro) activity.