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

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.

Keywords: SARS-CoV-2; allosteric inhibitors; drug development; native mass spectrometry; proteases.

Figure 1

Analysis of M^Pro by native MS. Left: Native mass spectra for M^Pro at different concentrations. Right: Representative plot of mole fraction versus concentration to quantify the dissociation constant and a view from an X‐ray structure of unligated M^Pro (PDB ID: 6YB7).

Figure 2

a) Native mass spectra of 5 μM M^Pro with the addition of different molar equivalents of x1187. To maintain similar solution conditions to the samples containing x1187, the control contains 10 % DMSO. b) Detailed view of dimer interface where x1187 binds (PDB ID: 5FRA).

Figure 3

a) Native mass spectrum for 5 μM M^Pro with 50 μM of the 11‐mer substrate at t=30 s. Peaks labelled TSAVLQ and +substrate indicate acyl‐enzyme complex and the non‐covalent enzyme‐substrate complex, respectively. b) mass spectra for the 15+ charge state at three representative times along the substrate cleavage reaction. Satellite peaks adjacent to the 15+ charge state are consistent with oxidation of between 4 and 8 of the ten methionine residues (+16 Da,). Peaks marked with asterisk corresponds to an impurity (+1042 Da). Inset: plot of the relative abundance of the enzyme‐substrate complex as a function of time. Solid line indicates the fit to a unimolecular kinetics model. c) Bar plot summarizing half‐lives of the enzyme‐substrate complex in the presence of different small molecules. Error bars represent standard deviation (n=3 independent replicates). *p<0.05, **p<0.001 (to M^Pro values). Representative mass spectra and kinetic plots for each dataset are shown in Figure S4.

Figure 4

Superimposition of views of x‐ray structures for unligated M^Pro (PDB 6YB7) and M^Pro bound to x1187 (PDB 5RFA), x0390 (PDB 5REC), x0464 (PDB 5REE), and x0425 (5RGJ).