Abstract
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.