Human Structural Homologues of SARS-CoV-2 PL pro as Anti-Targets: A Strategic Panel Analysis.

COVID-19 is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Developing small-molecule antiviral inhibitors of the SARS-CoV-2 papain-like protease (PL ^pro ) is advantageous due to the enzyme's role in processing viral polyproteins and disrupting host immune sensing. Given the structural and functional similarities between PL ^pro and human deubiquitinases (DUBs), small-molecule inhibitors are frequently counter-screened for off-target activity using a panel of human DUBs. Through X-ray crystallography, DALI structural comparisons, and in silico analysis, a high-quality crystal structure of SARS-CoV-2 PL ^pro enabled the identification of the closest structural human homologues of PL ^pro . Among the 27 human DUBs identified, USP46 and USP12 displayed the greatest structural similarity to PL ^pro , with alignment scores below 0.45 and RMSD values of 3.0 Å or less. Additionally, binding sites on ubiquitin-specific protease (USP46) and USP12, ancillary to the active site residues, share high sequence identity to the PL ^pro substrate binding sites that are often engaged by the most potent PL ^pro inhibitors. These findings offer a strong basis for choosing anti-targets and serve as a foundation for designing selective small-molecule PL ^pro inhibitors.