Active components in Ephedra sinica stapf disrupt the interaction between ACE2 and SARS-CoV-2 RBD: Potent COVID-19 therapeutic agents

ETHNOPHARMACOLOGICAL RELEVANCE: Ephedra sinica Stapf is a widely used folk medicine in Asia to treat lung diseases, such as cold, cough and asthma. Many efforts have revealed that some traditional Chinese medicine (TCM) prescriptions containing Ephedra sinica could effectively alleviate the symptoms and prevent the fatal deterioration of COVID-19. AIM OF THE STUDY: The present study aims to discover active compounds in Ephedra sinica disrupting the interaction between angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor-binding domain (SARS-CoV-2 RBD) to inhibit SARS-CoV-2 virus infection. MATERIALS AND METHODS: The ethanol extracts of Ephedra sinica were prepared. Activity guided isolation of constituents was carried out by measuring the inhibitory activity on ACE2-RBD interaction. The structures of active compounds were identified by HPLC-Q-TOF-MS/MS and NMR. To testify the contribution of main components for the inhibitory activity, different samples were prepared by components knock-out strategy. The mechanism of compounds inhibiting protein-protein interaction (PPI) was explored by competition inhibition assays, surface plasmon resonance (SPR) assays and molecular docking. SARS-CoV-2 S protein-pseudoviruses were used to observe the viropexis effect in cells. RESULTS: Ephedra sinica extracts (ESE) could effectively inhibit the interaction between ACE2 and SARS-CoV-2 RBD (IC(50) = 95.01 μg/mL). Three active compounds, 4,6-dihydroxyquinoline-2-carboxylic acid, 4-hydroxyquinoline-2-carboxylic acid and 4-hydroxy-6-methoxyquinoline-2-carboxylic acid were identified to inhibit ACE2-RBD interaction (IC(50) = 0.58 μM, 0.07 μM and 0.15 μM respectively). And knock-out the three components could eliminate the inhibitory activity of ESE. Molecular docking calculations indicated that the hydrogen bond was the major intermolecular force. Finally, our results also showed that these compounds could inhibit the infectivity of SARS-CoV-2 S protein-pseudoviruses to 293T-ACE2 (IC(50) = 0.44–1.09 μM) and Calu-3 cells. CONCLUSION: These findings suggested that quinoline-2-carboxylic acids in Ephedra sinica could be considered as potential therapeutic agents for COVID-19. Further, this study provided some justification for the ethnomedicinal use of Ephedra sinica for COVID-19.