Inhibitory potential of Morus alba leaf extract and its phytoconstituent against SARS-CoV-2 main protease: An integrative in silico and in vitro analysis.

The SARS-CoV-2 3CLpro is crucial for viral replication and is therefore recognized as a promising drug target. In this study, the 3CLpro gene was cloned and transiently expressed in CHO cells to establish a functional in vitro drug screening model. The inhibitory potential of the Morus alba (MA) leaves extract was evaluated. The MTT assay revealed that the extract was non-toxic to CHO cells at concentrations ranging from 3.12 to 100 μg/mL qPCR and Western blot analysis showed a significant downregulation of the 3CLpro gene with up to 99% inhibition at the mRNA level (IC50 = 105 ± 5 μg/mL), and up to 80% at the protein level at a 100 μg/mL dose. Twenty-nine Morus alba leaf compounds were computationally screened to identify the potential 3CLpro suppressors. Naringin, nicotiflorin, and cyclomorusin emerged as top hits with binding affinities ranging from -9.8 to -9.6 kcal/mol, compared to the reference drug, i.e., nirmatrelvir (-9.0 kcal/mol). MM-GBSA and molecular dynamics simulation analysis further supported naringin as the relatively stable complex, exhibiting the lowest binding free energy (-63.03 kcal/mol) among cyclomorusin and nicotiflorin. In conclusion, these findings suggest that MA extract notably downregulated the transient expression of the main protease in CHO cells, and computational analysis identifies naringin as a potential allosteric inhibitor. However, further investigations are required to confirm direct enzyme-inhibition activity and viral infection studies for future drug development.