3CL^pro of SARS-CoV-2 as a new target for bufadienolides: in silico and in vitro study

The rapid spread of SARS-CoV-2 and its widespread public health implications have highlighted the urgent need for effective antiviral therapies. A promising strategy is to investigate natural compounds that may inhibit the key viral targets. In this work, we demonstrated the anti-SARS-CoV-2 activity of six bufadienolides, including bufalin (A), arenobufagin (B), gamabufotalin (C), telocinobufagin (D), marinobufagin (E), and bufarenogin (F) from the venom of the Central Asian green toad, Bufo viridis. Molecular docking assays revealed that all A-F bufadienolides bind to key residues (Thr26, His41, Met49, Met161, and Gln189) in the catalytic pocket of 3-chymotrypsin-like cysteine protease (3CL^pro), an essential enzyme for viral replication and polyprotein processing. The stability of the protein-ligand complexes was then tested using molecular dynamics (MD) simulations, while the binding free energies were estimated using the umbrella sampling (US) technique. Compounds A (-49.8 ± 1.0 kJ/mol), C (-45.9 ± 1.8 kJ/mol), E (-45.6 ± 1.1 kJ/mol), and F (-45.8 ± 1.9 kJ/mol) had significantly higher binding affinities than compounds B (-14.6 ± 1.1 kJ/mol) and D (-10.3 ± 1.9 kJ/mol). In vitro enzymatic assays also confirmed these results, demonstrating that A and C exhibited potent inhibitory activity against 3CL^pro with IC₅₀ values of 1.37 µM and 2 µM, respectively, compared to the other bufadienolides; however, they were less active than the positive control GC376 (IC₅₀ = 0.27 µM). The experimental results are consistent with the computational observations. In silico ADME profiling also revealed good pharmacokinetic properties, indicating that bufadienolides A-F are lead compounds for further antiviral drug development. Taken together, our results support the hypothesis that bufadienolides are SARS-CoV-2 3CL^pro inhibitors and elucidate their mechanism of action, thereby laying the foundation for potential therapeutic advances against COVID-19.