NIH Research Festival
High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotypingto establish a cell-based SARS-CoV-2 entry assay that can be used in biosafety level 2 (BSL-2) laboratories. The pseudotypedparticles (PPs) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PPs to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. From a screening of 5,158 approved drugs and clinically trialed drug candidates, 7 active compounds were identified that inhibited the SARS-CoV-2-S PP entry. Of these compounds, six compounds were active against live SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotypedwith spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.
Scientific Focus Area: Microbiology and Infectious Diseases
This page was last updated on Monday, September 25, 2023