Allosteric Inhibition of the SARS‐CoV‐2 Main Protease: Insights from Mass Spectrometry Based Assays. (15th October 2020)

• Record Type: Journal Article
• 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.
• Is Part Of: Angewandte Chemie. Volume 132:Number 52(2020)
• Page Start: 23750
• Page End: 23754
• Publication Date: 2020-10-15
• Subjects: allosteric inhibitors -- drug development -- proteases -- native mass spectrometry -- SARS-CoV-2; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.202010316
• Languages: English
• ISSNs: 0044-8249

Abstract

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. Abstract : 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 M Pro protease activity. Several small molecules bind non‐covalently to M Pro, do not compete for the active site, and slow the processing of the substrate, providing a means for optimizing potential antiviral compounds.