Development of Cell-based Assays to Identify SARS-CoV-2 Protease Inhibitor

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Coronaviruses are positive-strand RNA viruses with exceptionally large genomes (27-32 kb). All coronaviruses encode the enzymes required for their replication in genes 1a and 1b. Genes 1a and 1b are translated as polyproteins; 1b is expressed by -1 frameshifting at the end of the 1a open reading frame. In both SARS-CoV and SARS-CoV-2, the 1a polyprotein contains two proteases, a papain-like (PL) and a 3-chymotrypsin-like (3CL) protease. Together, these proteases cleave polyprotein at 14 sites (3 for PL protease, 11 for 3CL protease), generating 16 nonstructural proteins (nsps). These proteins include viral enzymes required for RNA replication including RNA-dependent RNA polymerase. The RNA replication machinery not only generates more copies of the viral genome but also generates subgenomic RNAs that act as mRNAs for the translation of structural proteins. Thus, the PL and 3CL proteases are critical for viral replication, which make them excellent targets for antivirals. ___We will develop human cell-based assays that can be used as pipelines to identify inhibitors of the SARS-CoV-2 3CL protease. These assays will have advantages over current protein-based assays that are often affected by in vitro assay conditions and do not account for compound absorption and toxicity issues. Furthermore, these assays are designed to be used in BSL-2 containment, making them far more accessible than the existing BSL-3 replication-competent virus assays, which are not amenable to high-throughput platforms.