Allosteric Inhibition of the SARS‐CoV‐2 Main Protease: Insights from Mass Spectrometry Based Assays

The SARS‐CoV‐2 main protease (Mpro) cleaves along the two viral polypeptides to release non‐structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus‐2019 disease. Here, we used native mass spectrometry to characterize the func...

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Bibliographic Details
Published in:Angewandte Chemie 2020-12, Vol.132 (52), p.23750-23754
Main Authors: El‐Baba, Tarick J., Lutomski, Corinne A., Kantsadi, Anastassia L., Malla, Tika R., John, Tobias, Mikhailov, Victor, Bolla, Jani R., Schofield, Christopher J., Zitzmann, Nicole, Vakonakis, Ioannis, Robinson, Carol V.
Format: Article
Language:English
Subjects:
allosteric inhibitors
Allosteric properties
Antiviral agents
Chemistry
Coronaviruses
Crystal structure
Dimers
drug development
Information processing
Mass spectrometry
Mass spectroscopy
native mass spectrometry
Polypeptides
Protease
proteases
Proteinase
SARS-CoV-2
Scientific imaging
Severe acute respiratory syndrome
Severe acute respiratory syndrome coronavirus 2
Spectroscopy
Structural proteins
Substrates
Viral diseases
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Summary:The SARS‐CoV‐2 main protease (Mpro) cleaves along the two viral polypeptides to release non‐structural proteins required for viral replication. MPro 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 Mpro. Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd=0.14±0.03 μM, indicating MPro 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 MPro activity. The SARS‐CoV‐2 main protease monomer/dimer equilibrium was characterized using native mass spectrometry. An MS‐based kinetic assay that quantifies the changes in the amounts of enzyme–substrate complex with time was used to capture MPro protease activity. Several small molecules bind non‐covalently to MPro, do not compete for the active site, and slow the processing of the substrate, providing a means for optimizing potential antiviral compounds.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.202010316