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.

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its...

A high-throughput mass spectrometry based Mpro assay identifies penicillin esters as new SARS-CoV-2 Mpro inhibitors.

Herein we provide a living summary of the data generated during the COVID Moonshot project focused on the development of SARS-CoV-2 main protease (Mpro) inhibitors. Our approach uniquely combines crowdsourced medicinal chemistry insights with high throughput crystallography, exascale computational chemistry infrastructure for simulations, and machine learning in triaging designs and predicting synthetic routes. This manuscript describes our methodologies leading to both covalent and non-covalent inhibitors displaying protease IC50 values under 150 nM and viral inhibition under 5 uM in multiple different viral replication assays. Furthermore, we provide over 200 crystal structures of fragment-like and lead-like molecules in complex with the main protease. Over 1000 synthesized and ordered compounds are also reported with the corresponding activity in Mpro enzymatic assays using two different experimental setups. The data referenced in this document will be continually updated to reflect the current experimental progress of the COVID Moonshot project, and serves as a citable reference for ensuing publications. All of the generated data is open to other researchers who may find it of use.

Inhibitors targeting the conserved nucleophilic cysteine of the mycobacterial l,d-transpeptidases are a potential strategy for the treatment of tuberculosis.

The SARS-CoV-2 main protease (M pro ) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent M pro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as M pro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.

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The two SARS‐CoV‐2 proteases, i. e . the main protease (M pro ) and the papain‐like protease (PL pro ), which hydrolyze the viral polypeptide chain giving functional non‐structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high‐throughput mass spectrometry (MS)‐based assay which directly monitors PL pro catalysis in vitro . The assay was applied to investigate the effect of reported small‐molecule PL pro inhibitors and selected M pro inhibitors on PL pro catalysis. The results reveal that some, but not all, PL pro inhibitor potencies differ substantially from those obtained using fluorescence‐based assays. Some substrate‐competing M pro inhibitors, notably PF‐07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL pro . Less selective M pro inhibitors, e. g . auranofin, inhibit PL pro , highlighting the potential for dual PL pro /M pro inhibition. MS‐based PL pro assays, which are orthogonal to widely employed fluorescence‐based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non‐covalent mechanisms.