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The emergence of the COVID-19 situation has become a global issue due to the lack of effective antiviral drugs for treatment. Flavonoids are a class of plant secondary metabolites that have antiviral activity against SARS-CoV-2 through inhibition of the main protease (3CL pro ). In this study, 22 flavonoids obtained from natural sources and semisynthetic approaches were investigated for their inhibitory activity against SARS-CoV-2 3CL pro , along with cytotoxicity on Vero cells. The protein-ligand interactions were examined using molecular dynamics simulation. Moreover, QSAR analysis was conducted to clarify the structural effects on bioactivity. Accordingly, the in vitro investigation demonstrated that four flavonoids, namely, tectochrysin ( 7 ), 6″,6″-dimethylchromeno-[2″,3″:7,8]-flavone ( 9 ), panduratin A ( 19 ), and genistein ( 20 ), showed higher protease inhibitory activity compared to the standard flavonoid baicalein. Finally, our finding suggests that genistein ( 20 ), an isoflavone discovered in Millettia brandisiana , has potential for further development as a SARS-CoV-2 3CL pro inhibitor.

The emergence of SARS-CoV-2 in December 2019 has become a global issue due to the continuous upsurge in patients and the lack of drug efficacy for treatment. SARS-CoV-2 3CL Pro is one of the most intriguing biomolecular targets among scientists worldwide for developing antiviral drugs due to its relevance in viral replication and transcription. Herein, we utilized computer-assisted drug screening to investigate 326 natural products from Thai traditional plants using structure-based virtual screening against SARS-CoV-2 3CL Pro . Following the virtual screening, the top 15 compounds based on binding energy and their interactions with key amino acid Cys145 were obtained. Subsequently, they were further evaluated for protein-ligand complex stability via molecular dynamics simulation and binding free energy calculation using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches. Following drug-likeness and ADME/Tox assessments, seven bisbenzylisoquinolines were obtained, including neferine ( 3 ), liensinine ( 4 ), isoliensinine ( 5 ), dinklacorine ( 8 ), tiliacorinine ( 13 ), 2'-nortiliacorinine ( 14 ), and yanangcorinine ( 15 ). These compounds computationally showed a higher binding affinity than native N3 and GC-373 inhibitors and attained stable interactions on the active site of 3CL pro during 100 ns in molecular dynamics (MD) simulation. Moreover, the in vitro enzymatic assay showed that most bisbenzylisoquinolines could experimentally inhibit SARS-CoV-2 3CL Pro . To our delight, isoliensinine ( 5 ) isolated from Nelumbo nucifera demonstrated the highest inhibition of protease activity with the IC 50 value of 29.93 μM with low toxicity on Vero cells. Our findings suggested that bisbenzylisoquinoline scaffolds could be potentially used as an in vivo model for the development of effective anti-SARS-CoV-2 drugs.