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

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.


Protein production and purification
The plasmid encoding the codon-optimized gene for SARS-CoV-2 M Pro fused with an N-terminal glutathione S-transferase tag and a C-terminal His6-tag preceded by a human rhinovirus 3C protease site was transformed into E. coli Rosetta (DE3). Several transformed clones were used to inoculate a starter culture which was grown to exponential phase in lysogeny broth at 37 °C containing 100 μg mL -1 carbenicillin. The starter culture was used to inoculate 1 L of Terrific Broth Autoinduction media (Formedium) supplemented with 10% (v/v) glucose and 100 μg mL -1 carbenicillin. Cells were grown for 5 h at 37 °C, then cooled to 18 °C and grown for an additional 12 h for recombinant protein production. Cells were harvested by centrifugation (5,000 x g, 15 min).
Cells were resuspended in lysis buffer consisting of 50 mM Tris-HCl (pH 8), 300 mM NaCl, 10 mM imidazole and 0.05 mg/mL benzonase (Sigma Aldrich) and lysed by sonication. Lysates were clarified by centrifugation (50,000 x g, 1 hr). The supernatant was loaded onto a HiTrap Talon Co 2+ affinity column (GE Healthcare) equilibrated with lysis buffer. The column was washed with lysis buffer supplemented with 25 mM imidazole. Elution was performed with 50 mM Tris pH 8.0, 300 mM NaCl and 500 mM imidazole followed by His6-tag cleavage using human rhinovirus (HRV) 3C protease (produced in-house) at 4°C. Reverse immobilized metal affinity chromatography was used to remove the HRV 3C protease and His6-tag. The flow-through was concentrated and loaded onto Superdex 75 26/600 size exclusion chromatography column equilibrated with 50 mM Tris-HCl pH 8 and 300 mM NaCl. Protein purity was assessed at each step by SDS-PAGE. Peak fractions for dimeric M Pro were pooled and concentrated before mass spectrometry (MS) analysis.

Sample preparation
Prior to mass spectrometry (MS) analysis, fresh aliquots of M Pro were buffer exchanged into 200 mM ammonium acetate (pH 7.4) using Zeba spin 7k buffer exchange columns (Thermo Scientific). Fragments were taken from the DSI-poised library as 10 mM stocks in 100% DMSO, diluted with 200 mM ammonium acetate (pH 7.4), and mixed with M Pro in appropriate amounts prior to analysis. For kinetic measurements, 50 µM of 11-mer substrate was mixed with 5 µM of M Pro and analyzed immediately. The inclusion the fragments as well as the 11-mer substrate incorporates 10% DMSO into M Pro containing solutions for MS analysis. Therefore, we have included 10% DMSO in all drug binding and kinetics experiments. All measurements were performed in triplicate.

Native mass spectrometry
1-3 µL of protein solution was loaded into in-house prepared gold-coated electrospray capillaries pulled to ~1-3 µm tip diameter. [2] Samples were analyzed using a Thermo Q-Exactive UHMR Orbitrap platform operated at 30,000 resolving power (at m/z 200). An electrospray was generated by applying 0.9 -1.3 kV bias to the electrospray capillary; no backing pressure was applied to the electrospray solution. The instrument capillary was maintained at a temperature of ~100 °C to assist with desolvation. The instrument was operated at a resolving power of 12,500 (at m/z 200) throughout this study using manufacturer recommended instrument settings for native MS, and N2 as the background gas (pressure setting of 8). No collision energy was applied throughout the instrument.

Data Analysis
To extract the relative abundance of the different species in each spectrum, mass spectral data were deconvoluted to zero-charge spectra using Unidec software. [3] Mole fraction for monomer and dimer ratio were calculated at each concentration and fitted to a monomer-dimer binding model described by Bergdoll et al. [4] Kinetic models were fitted to the experimental data using [ ] = [ ] 0 * − where [ES]t and [ES]0 are the quantities of enzyme-substrate determined at time t = t and t = 0, respectively, and k is the measured rate constant (in s -1 ). All data models were fit to experimental data sets using a user-defined function in OriginPro 2018 (OriginLab Corporation, Northampton, MA) using a least-squares residual analysis. Figure S1. Effect of x0464, x0425, and x0390 on M Pro monomer-dimer equilibria. No substantial differences were observed in the peaks assigned to monomer (10 + ) and dimer (15 + ). Figure S2. Expansion of the charge state series assigned to monomeric M pro in the presence of 50 mM of the 11-mer substrate 30s after addition of the substrate. The expected peak series for M Pro monomers bound to the 11-mer substrate (indicated with red asterisk) is not observed. Figure S3. MS data collected for the 11-mer substrate in 200 mM ammonium acetate (pH 7.4) at ~10 µM (left) and the products formed upon M Pro cleavage of the 11-mer substrate at t = 570 s to form two peptide products (right).  Figure S5. Covalent modification of Cys145 greatly diminishes M Pro proteolytic activity. 5 µM M Pro was mixed with 100 µM IPA3 (20-fold excess prepared in 100% DMSO) and incubated for 15 min. Mass spectra were collected immediately following the addition of 50 µM 11-mer substrate. (a) Native mass spectra collected at several different timepoints showing highly modified M Pro monomers and dimers; no signals for enzymesubstrate complexes were observed. Inset shows extent of covalent modification by IPA3 (+175 Da per modification). (b) Deconvoluted mass spectra collected under instrument conditions that preferentially transmit low mass-to-charge (m/z) species. At t = 30 min, the dominant feature is the 11-mer substratelow intensity peaks assigned to the products (m/z 593 and m/z 617) are observed.