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 M pro cleaves at eleven sites to release non‐structural proteins required for viral replication. M Pro 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 M pro . Analysis of the monomer‐dimer equilibria reveals a dissociation constant of K d = 0.14 ± 0.03 µM, revealing M Pro 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 M Pro activity.

Computational analysis of dynamic allostery and control in the SARS-CoV-2 main protease

“…Computational studies such as this, therefore, accompany and support concurrent experimental programmes of scanning for small-molecule binding candidates to the protein. We note that several candidate molecules, identified in very recent large crystallographic fragment screens against SARS-CoV-2 M pro [31,32], bind to regions suggested as dynamically sensitive control candidates in this study. Such experimental programmes are essential in identifying inhibitors that are both effective, and have sufficient total binding free energies to the protein.…”

Allosteric Hotspots in the Main Protease of SARS-CoV-2

“…Using the above described approaches we have identified four allosteric binding sites on the protease. We describe the location of the sites and been shown to exhibit some inhibitory effect on the M pro in a recent study [24]. Notably, site 2, although not directly coupled to the active site as a functional site, is located in the dimer interface ( We also include the analysis of 15 structures containing small fragments from a recent Diamond Light Source XChem fragment screen [50] which bind in proximity to the putative sites.…”

“…The active site score is analysed rigorously with a structural bootstrap to compare the effect of each fragment on the protease. Some fragments have a direct link to the active site and have been recently investigated in experimental studies [24] and might provide a first starting point for rational drug design.…”

“…We subsequently scored the active sites in each run (full data in Table S9) and found that the fragment deposited with the PDB identifier 5RE8 might be of particular interest as it has the highest connectivity to the active site. Moreover, one of the fragments within 4 Å of site 1 with the PDB identifier 5RGJ, has been shown to inhibit the proteolytic activity of the M pro [24] and possesses a relatively high connectivity to the active site.…”

“…(For reviews and recent successes see Wenthur et al and Cimermancic et al [22,23]). To the best of our knowledge, there is to date no indication of such putative allosteric sites of the coronavirus M pro s in the literature other than a recent implication of potential allosteric regulation of SARS-CoV-2 M pro [24] and simulated binding events to distant areas of the protein [25]. Encouragingly, however, there have been indications of allosteric processes mediated by the extra domain in the SARS-CoV M pro [26,27,28,14].…”