A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Abstract

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. 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 pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) 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 PP 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. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating 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 pseudotyped with 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.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics

“…HEK-293T cells overexpressing human ACE2 47 were obtained from BEI Resources and maintained in DMEM as above. SARS-CoV-2 pseudovirus particles encoding for luciferase expression were prepared using the plasmids and protocols essentially as previously described 48 . The pseudoviral infectivity assay was also similar to previous publications 48 .…”

“…SARS-CoV-2 pseudovirus particles encoding for luciferase expression were prepared using the plasmids and protocols essentially as previously described 48 . The pseudoviral infectivity assay was also similar to previous publications 48 . Briefly, HEK293-ACE2 cells were seeded in 384-well white plates (PerkinElmer, USA), allowed to attach overnight followed by addition of pseudoviral particles in the presence or absence of varying concentrations of GRFT.…”