A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors

Yin, Xinjian; Chen, Litong; Yuan, Siwen; Liu, Lan; Gao, Zhizeng

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC(50) of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal C) were measured to be 6 nM, 2.5 nM, and 8.9 µM respectively. In addition, we found Sal A has a slightly lower IC(50) (3.9 µM) than Sal C. Interestingly, simple caffeic acid can also disrupt the formation of 6-HB with a sub-mM concentration. Pilot high throughput screening (HTS) of a small marine natural product library validates the assay with a Z’ factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitors and assess their binding affinity.