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

Tarick J El‐Baba,Anastassia L Kantsadi,Tobias John,Nicole Zitzmann,Ioannis Vakonakis,Carol V Robinson,Jani R Bolla,Christopher J Schofield,Corinne A Lutomski,Victor Mikhailov,Tika R Malla

doi:10.1002/anie.202010316

Tarick J El‐Baba, Anastassia L Kantsadi + Show 9 more

Open Access

https://doi.org/10.1002/anie.202010316

Copy DOI

Abstract

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 K d=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.

Highlights

The SARS-CoV-2 main protease (Mpro) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication
We report the dissociation constant for SARS-CoV-2 MPro determined using native mass spectrometry (MS), which can directly identify and quantify the relative amounts of the oligomeric state of a protein in solution.[6]
As the concentration of protein is decreased in a series of stepwise dilutions from 5.0 mM to 0.625 mM, the signals corresponding to the MPro monomer increase concurrent with a decrease in the peak intensities assigned to the dimer

Summary

Introduction

The SARS-CoV-2 main protease (Mpro) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. The high-abundant signals centered at the 17+ charge state correspond to a deconvoluted mass of 67 591 Æ 0.5 Da, consistent with the expected sequence mass of dimeric MPro (67 592 Da). As the concentration of protein is decreased in a series of stepwise dilutions from 5.0 mM to 0.625 mM, the signals corresponding to the MPro monomer increase concurrent with a decrease in the peak intensities assigned to the dimer.

Results

Conclusion