Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays

Abstract

Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro 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 Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 ± 0.03 μM, revealing MPro 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 MPro activity.

Proteomics‐based mass spectrometry profiling of SARS‐CoV‐2 infection from human nasopharyngeal samples

“…In another study, El‐baba et al investigated the SARS‐CoV‐2 main protease known to play a role in viral replication. Monomer/dimer equilibrium was analyzed in the presence of several small molecules that non‐covalently bound to the protease slowing down the substrate processing, thereby optimizing the use of antiviral compounds in this context (El‐Baba et al, 2020). Native and denaturing top‐down MS‐based approaches to determine the glycosylation of the SARS‐CoV‐2 S‐protein and its interaction with the ACE2 receptor is described in Section 3.4.…”

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

“…2D and 6) and suggesting that nsp7-11 binding stabilizes the Mpro dimer. In this sense, El-Baba and colleagues (61) identified that fragment JGY-discovered through crystallographic fragment screening and binding in the dimer interface (30)-destabilized the Mpro dimer and showed ~35% inhibition of the rate of processing at 100 μM. Along the same lines, Sun and colleagues (62) discovered a nanobody, NB2B4, which binds the C-terminal domain of monomeric Mpro (PDB 7VFB) and inhibits activity with an IC 50 ~150 nM.…”

Mass Spectrometry-Based Structural Virology

“…Work of Robinson and Vakonakis 192 focused on the protease M encoded by the SARS-COV-2 RNA genome. Its role is to process several of the virus structural proteins.…”