A High-throughput screening system for SARS-CoV-2 entry inhibition, syncytia formation and cell toxicity 

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to host cell is mediated through the binding of the SARS-CoV-2 Spike protein via receptor binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2). Identifying compounds inhibiting Spike-ACE2 binding would be a promising, safe antiviral approach against COVID-19. Methods: In the present study, we have used BSL-2 compatible replication-competent vesicular stomatitis virus (VSV) replaced glycoprotein with spike protein of SARS-CoV-2 expressing eGFP reporter system (VSV-eGFP-SARS-CoV2) in a permissive cells harboring cytotoxicity marker. The high-throughput compatible SARS-CoV-2 permissive reporter system that encompasses cells stably expressing hACE2 tagged cerulean and nuclear H2B tagged with mCherry, as a marker of nuclear condensation that also enabled imaging of fused cells among infected EGFP positive cells and could give real-time information of syncytia formation. Results: A limited high-throughput screening identified six natural products with marked VSV-eGFP-SARS-CoV2 inhibition at non cytotoxic dose. Molecular simulation studies with positive hits in complex with wild-type spike reaffirm their potential to impede viral entry. Real-time syncytia formation assay of the molecules revealed inhibition of syncytia with Didemnin B, and delayed inhibition with other natural products such as Scillaren A, Proscillaridin, Acetoxycycloheximide indicating that the assay is a reliable platform for any image based drug screening. Conclusion: BSL-2 compatible assay system equivalent to the infectious SARS-CoV-2 is a promising tool for high-throughput screening of large compound libraries for viral entry inhibitors against SARS-CoV-2 along with toxicity and effect on syncytia. Studies using clinical isolates of SARS-CoV-2 is warranted to confirm the antiviral potency of the leads and the utility of the screening system.