A robust high-throughput fluorescent polarization assay for the evaluation and screening of SARS-CoV-2 fusion inhibitors.

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 IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal-C) were measured to be 6.1 nM, 2.5 nM, and 8.9 µM respectively. In addition, we found Sal-A has a slightly lower IC50 (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.

Three salvianolic acids inhibit 2019-nCoV spike pseudovirus viropexis by binding to both its RBD and receptor ACE2.

Since December 2019, the new coronavirus (also known as severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, 2019-nCoV])-induced disease, COVID-19, has spread rapidly worldwide. Studies have reported that the traditional Chinese medicine Salvia miltiorrhiza possesses remarkable antiviral properties; however, the anti-coronaviral activity of its main components, salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) is still debated. In this study, we used Cell Counting Kit-8 staining and flow cytometry to evaluate the toxicity of SAA, SAB, and SAC on ACE2 (angiotensin-converting enzyme 2) high-expressing HEK293T cells (ACE2h cells). We found that SAA, SAB, and SAC had a minor effect on the viability of ACE2h cells at concentrations below 100 µM. We further evaluated the binding capacity of SAA, SAB, and SAC to ACE2 and the spike protein of 2019-nCoV using molecular docking and surface plasmon resonance. They could bind to the receptor-binding domain (RBD) of the 2019-nCoV with a binding constant (KD ) of (3.82 ± 0.43) e-6 M, (5.15 ± 0.64)e-7 M, and (2.19 ± 0.14)e-6 M; and bind to ACE2 with KD (4.08 ± 0.61)e-7 M, (2.95 ± 0.78)e-7 M, and (7.32 ± 0.42)e-7 M, respectively. As a result, SAA, SAB, and SAC were determined to inhibit the entry of 2019-nCoV Spike pseudovirus with an EC50 of 11.31, 6.22, and 10.14 µM on ACE2h cells, respectively. In conclusion, our study revealed that three Salvianolic acids can inhibit the entry of 2019-nCoV spike pseudovirus into ACE2h cells by binding to the RBD of the 2019-nCoV spike protein and ACE2 protein.