Identification of SARS-CoV-2 3CLpro inhibitors from marine actinomycetes through integrated phylogeny-based metabolomics with functional screening and bioinformatic analysis

As the acute threat of the COVID-19 pandemic diminishes globally, advancing the research and development of antiviral agents remains crucial for ensuring preparedness for future outbreaks. This study investigates the inhibitory effects of marine sediment-derived actinomycetes on the SARS-CoV-2 main protease (3CLpro), a key enzyme involved in viral replication and transcription. Extracts of 75 actinomycete strains from two locations, Nogas Island in Antique and Tubbataha Reefs Natural Park in Palawan, Philippines, were evaluated for their ability to inhibit 3CLpro, with 36% of the extracts showing more than 90% inhibition. Further examination of 27 extracts revealed IC₅₀ values between 2 and 8 µg/mL, suggesting notable inhibitory strength. Phylogenetic analysis grouped the active strains into seven distinct clades, with Clades 1, 5, and 6, closely related to Streptomyces enissocaesilis, S. geysiriensis, S. ardesiacus, and S. indicus, exhibiting antiviral activity. Metabolomic profiling using LCMS-QTOF, coupled with principal component analysis (PCA) and hierarchical clustering, revealed that metabolic signatures were consistent with clade classification. Shared features among the active clades included a biohopanoid, fatty acid amides, diketopiperazines, lipid derivatives, and aromatic compounds. Supervised analysis using orthogonal partial least squares discriminant analysis (OPLS-DA) identified 17 discriminant metabolites produced by Streptomyces strains associated with antiviral activity, including a bicyclic sesquiterpenoid, an aromatic polyketide, an alkylphenol, fatty acid derivatives, alkylated aromatic amines, lipid-derived metabolites, and a carbazole alkaloid. Among the identified compounds, streptocarbazole C showed a strong predicted binding affinity to SARS-CoV-2 3CLpro (- 9.8 ± 0.30 kcal/mol) in molecular docking analysis, with favorable interactions at the enzyme's catalytic dyad (HIS41 and CYS145). In silico toxicity predictions suggested a generally favorable safety profile, although some limitations were noted. These findings highlight the biosynthetic and functional potential of marine sediment-derived Streptomyces against SARS-CoV-2 3CLpro, providing a framework for the discovery of antiviral natural products through the integration of ecological, phylogenetic, metabolomics, and computational analyses.