2020-144 Small Molecule Inhibitors of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as COVID-19, is a human betacoronavirus that encodes for a papain-like protease (PLpro) that is responsible for maturation of the CoV viral poly-peptide as well as suppression of host innate immune responses. Inhibition of the host innate immune response is through the reversal of post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). We have characterized the biochemical activity of SARS-CoV-2 Plpro on ubiquitin and ISG15 substrates as well as identifying small molecule inhibitors of the enzyme function. Modification of host proteins by Ub and ISG15 has been shown to facilitate 1) the NFkB inflammation and IFN-I responses, 2) upregulation of the production of cytokines, chemokines, and other IFN-stimulated gene products with antiviral properties as well as 3) the degradation or sequestration of viral proteins. Differences in enzyme substrate specificity have been shown for the eight different linkage forms of polyubiquitin. PLpro activity is also sensitive to species-species variations within ISG15. The dual viral polypeptide cleavage and immune suppression roles of PLpro makes the enzyme an interesting target for small molecule antiviral development. We have shown the first biochemical characterization of the deubiquitinating and deISGylating activities of the SARS-CoV-2 PLpro using 7-amino-4-methyl coumarin conjugated Ub and ISG15. We further reveal SARS-CoV-2 PLpro’s lack of deubiquitinase activity toward a monomeric Ub substrate, and explore the protease’s ability to cleave the eight different poly-Ub linkages. We have characterized the naphthalene based PLpro inhibitor activity in SARS-CoV-2 that were originally designed for SARS-CoV infection. Cory Acuff cacuff@uga.edu 706-542-5682