Modified Project Summary/Abstract Section SARS-CoV-2, belonging to the genus betacoronavirus, encodes two large overlapping polyprotein precursors (pp1a and pp1ab), four structural proteins (spike, envelope, membrane, and nucleocapsid), and several accessory proteins. The two polyproteins (pp1a/pp1ab) must be cleaved into their individual, nonstructural proteins for successful viral replication (1). Two viral proteases are essential and responsible for processing the polyproteins: the 3C-like protease (3CL protease; CLpro also referred to as “main protease”, Mpro) and a papain-like protease (PLpro) (2). Importantly, CLpro cleaves polypeptides after a glutamine residue in the P1 position of the substrate, which is a unique activity not observed in other human proteases and suggests that this viral protease can be specifically and selectively inhibited by a small molecule inhibitor (3). PLpro also suppresses the innate immune response by removing interfernon-stimulated gene 15 (ISG) from viral and host proteins. Therefore, both CLpro and PLpro are important direct-acting targets for oral anti-viral development. We will develop drug candidates for each of these targets, from early discovery to late lead optimization. In the first aim, we have a non-covalent CLpro where we are in hit to lead chemistry and have generated a suitable compound for PK studies to potentially test for in vivo efficacy testing. In the second aim we are initiating formal hit assessment on PLpro hit compounds based on HCV drugs and other chemical templates with the goal in the end of year 1 to enter formal hit to lead chemistry given the overall challenge in the field of drugging this target. Additionally, we plan on screening for novel PLpro and CLpro using novel chemical libraries at Scripps using NMR screening and cell-based reporter assays. This project attempts to have a balanced portfolio between late-stage preclinical drug candidates and novel approaches to these two essential viral drug targets.