In Silico Discovery and Experimental Validation of Novel Small Molecule Inhibitors Targeting SARS-CoV-2 Main Protease (Mpro) from Marine Natural Products

The persistent emergence of SARS-CoV-2 variants highlights an ongoing need for structurally diverse antiviral therapeutics. The SARS-CoV-2 main protease (Mpro), essential for viral polyprotein processing and replication, represents a validated target with high evolutionary conservation. In this study, we implemented an integrated workflow combining structure-based virtual screening, molecular dynamics (MD) simulations, binding free energy calculations, and in vitro enzymatic validation to identify novel Mpro inhibitors from a marine natural product repository. Computational screening of 5,420 marine-derived metabolites against the catalytic dyad (His41-Cys145) identified two distinct scaffolds—a brominated sesquiterpenoid hydroquinone (MNP-412) and a substituted spongian diterpenoid (MNP-883)—exhibiting superior binding affinity over standard reference ligands. All-atom 200 ns MD simulations and MM-GBSA free energy decomposition revealed persistent key hydrogen-bonding networks with residues Glu166, Gln189, and Cys145, accompanied by stable conformational trajectories (RMSD < 1.8 Å). In vitro fluorescence resonance energy transfer (FRET) enzyme inhibition assays confirmed that MNP-412 and MNP-883 inhibited recombinant SARS-CoV-2 Mpro with IC₅₀ values of 2.14 ± 0.18 µM and 4.76 ± 0.35 µM, respectively. Furthermore, cellular cytotoxicity assays demonstrated low cytotoxicity in Vero E6 cells (CC₅₀ > 100 µM), yielding high selectivity indices. These findings establish marine secondary metabolites as promising non-peptidic leads for the rational development of next-generation coronaviral therapeutics.