Effect of dihydromyricetin on SARS-CoV-2 viral replication and pulmonary inflammation and fibrosis

Background: COVID-19 (Coronavirus Disease-2019) has spread widely around the world and impacted human health for millions. The lack of effective targeted drugs and vaccines forces scientific world to search for new effective antiviral therapeutic drugs. It has reported that flavonoids have potential inhibitory activity on SARSCoV-2 M-pro and anti-inflammatory properties. Dihydromyricetin, as a flavonol, also has antiviral and antiinflammatory potential. However, the inhibition of dihydromyricetin on SARS-CoV-2 M-pro and the protective effect of dihydromyricetin on pulmonary inflammation and fibrosis have not been proved and explained. Purpose: The coronavirus main protease (M-pro) is essential for SARS-CoV-2 replication and to be recognized as an attractive drug target, we expect to find the inhibitor of M-pro. Novel coronavirus infection can cause severe inflammation and even sequelae of pulmonary fibrosis in critically ill patients. We hope to find a drug that can not only inhibit virus replication but also alleviate inflammation and pulmonary fibrosis in patients. Methods: FRET-based enzymatic assay was used to evaluate the inhibit activity of dihydromyricetin on SARSCoV-2 M-pro. Molecular docking was used to identify the binding pose of dihydromyricetin with SARS-CoV-2 M-pro. The protective effects of dihydromyricetin against BLM-induced pulmonary inflammation and fibrosis were investigated in C57BL6 mice. BALF and lung tissue were collected for inflammation cells count, ELISA, masson and HE staining, western blotting and immunohistochemistry to analyze the effects of dihydromyricetin on pulmonary inflammation and fibrosis. MTT, western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and wound healing were used to analyze the effects of dihydromyricetin on lung fibrosis mechanisms in Mlg cells. Results: In this study, we found that dihydromyricetin is a potent inhibitor targeting the SARS-CoV-2 M-pro with a half-maximum inhibitory concentration (IC50) of 1.716 +/- 0.419 mu M, using molecular docking and the FRETbased enzymatic assay. The binding pose of dihydromyricetin with SARS-CoV-2 M-pro was identified using molecular docking method. In the binding pocket of SARS-CoV-2 M-pro, the dihydrochromone ring of dihydromyricetin interact with the imidazole side chain of His163 through pi-pi stacking. The 1-oxygen of dihydromyricetin forms a hydrogen bond with the backbone nitrogen of Glu166. The 3-, 7-, 3'- and 4'-hydroxyl of dihydromyricetin interact with Gln189, Leu141, Arg188 and Thr190 through hydrogen bonds. Moreover, our results showed that dihydromyricetin can significantly alleviate BLM-induced pulmonary inflammation by inhibiting the infiltration of inflammation cells and the secretion of inflammation factors in the early process and also ameliorate pulmonary fibrosis by improving pulmonary function and down-regulate the expression of alpha-SMA and fibronectin in vivo. Our results also showed that dihydromyricetin inhibits the migration and activation of myofibroblasts and extracellular matrix production via transforming growth factor (TGF)-beta 1/Smad signaling pathways. Conclusion: Dihydromyricetin is an effective inhibitor for SARS-CoV-2 M-pro and it prevents BLM-induced pulmonary inflammation and fibrosis in mice. Dihydromyricetin will be a potential medicine for the treatment of COVID-19 and its sequelae.