Creation and Validation of cell-based screening systems for SARS-CoV-2 drug targets

Project Summary/Abstract: Effective antivirals are sorely needed against SARS CoV-2 (CoV2), and coronaviruses (CoV) in general. Screening systems for drugs targeting CoV2 enzymes usually involve screening against purified enzymes. This can be technically challenging, and the vast majority of inhibitors identified ultimately lack utility as antivirals because: they may not be transported into and retained in cells, altered within cells such that they no longer inhibit the target effectively, or exhibit off-target effects that preclude their use as therapeutics. Using predicted drugs directly in viral infection assays can exhibit off-target effects against the host cell or other viral proteins and can be difficult to distinguish within the context of the viral infection (the inhibition of CoV2 infection by hydroxychloroquine is one such example). Ideally screens for viral enzymes isolate the enzyme from the viral infection, but evaluate function within human cells. We have constructed vectors for expression of four wild-type CoV2 proteins, and unique biosensors, to evaluate function of two CoV2 enzymatic functions, the CoV Main Protease and the CoV RNA-dependent RNA polymerase complex. We are completing assay validation and beginning to use the assays to evaluate the few known inhibitors of these enzymes. The protease assay will be converted to a stable cell line for large scale screening programs. The transfection assays will be evaluated using both established and experimental 3CL and RdRpC inhibitors, and 128 FDA- approved drugs that we have predicted target these enzymes, to validate the usefulness of these screening assays. Drugs that show inhibition of activity will be quantified using dose-response analysis in the cell-based assays, and subsequently analyzed for inhibition of CoV2 infection of cultured human lung cells across a similar dose-response range. Following validation, vectors and/or stable cells lines for the cell-based drug screening systems will be made readily available to other researchers, such as NIH's NCATS, who are currently seeking CoV antiviral agents.