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The 3C-like protease (3CL pro ) is crucial to the replication of SARS-CoV-2, the causative agent of COVID-19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CL pro only functions as a homodimer, making the protein-protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS-CoV-2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration-dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600-fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein-protein interaction inhibitors targeting 3CL pro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CL pro enzymes are capable of replication., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Drugs that act by covalently attaching to their targets have been used to treat human diseases for over a hundred years. However, the deliberate design of covalent drugs was discouraged due to concerns of toxicity and off-target effects. Recent successes in covalent drug discovery have sparked fresh interest in this field. New screening and testing methods aimed at covalent inhibitors can play pivotal roles in facilitating the discovery process. This feature article focuses on computational and biophysical advances originating from our labs over the past decade and how these approaches have contributed to the design of prolyl oligopeptidase (POP) and SARS-CoV-2 3CL pro covalent inhibitors.

The 3C‐like protease (3CLpro) is crucial to the replication of SARS‐CoV‐2, the causative agent of COVID‐19, and is the target of several successful drugs including Paxlovid and Xocova. Nevertheless, the emergence of viral resistance underlines the need for alternative drug strategies. 3CLpro only functions as a homodimer, making the protein–protein interface an attractive drug target. Dimerization is partly mediated by a conserved glycine at position 11. However, some naturally occurring SARS‐CoV‐2 sequences contain a serine at this position, potentially disrupting the dimer. We have used concentration‐dependent activity assays and mass spectrometry to show that indeed the G11S mutation reduces the stability of the dimer by 600‐fold. This helps to set a quantitative benchmark for the minimum potency required of any future protein–protein interaction inhibitors targeting 3CLpro and raises interesting questions regarding how coronaviruses bearing such weakly dimerizing 3CLpro enzymes are capable of replication. [ABSTRACT FROM AUTHOR]