Marine Streptomyces-Derived Lipids Inhibit SARS-CoV-2 3CLpro Through In Vitro and Predicted Multi-Site Binding Mechanisms.

Background: The SARS-CoV-2 3CLpro is essential for viral replication and an attractive target for antiviral intervention. While most strategies target the catalytic site, recent studies suggest that the dimerization interface and cryptic allosteric pockets offer alternative mechanisms for inhibition. Objective: This study investigated lipid metabolites from the marine sediment-derived Streptomyces sp. DSD454T as potential multi-site 3CLpro inhibitors. Methods: Metabolites were extracted from cultured biomass and characterized using LCMS-QTOF, MS/MS (LCMS-TQ), and 1H NMR, with identities confirmed against authentic standards. 3CLpro inhibition was assessed using a FRET-based assay, and ligand-protein interactions were evaluated through molecular docking and MM/GBSA calculations. Lipid content and comparative lipidomic signatures were examined across bioactive Streptomyces strains through LCMS-TQ and BODIPYTM 493/503 staining. Results: Palmitoleic and linoleic acids were identified as major constituents and inhibited SARS-CoV-2 3CLpro with IC50 values of 1.59 µg/mL (6.25 µM) and 5.29 µg/mL (18.88 µM). Molecular docking predicted that both fatty acids bind not only to the catalytic site but also to the dimerization interface and cryptic allosteric pocket. Additional lipids, including 9-heptadecenoic acid, linolenic acid, 9-HODE, and monoacylglycerols such as aggrecerides A-C and glyceryl-based lipids, showed similarly favorable multi-site binding profiles. Streptomyces sp. DSD454T also exhibited substantial lipid accumulation (~63% of crude extract). Across bioactive Streptomyces strains, a conserved lipid signature correlated strongly with 3CLpro inhibition. Conclusions: This study highlights the potential of microbial lipids as promising scaffolds for developing catalytic and allosteric SARS-CoV-2 3CLpro inhibitors and underscore marine Streptomyces as a valuable source of structurally simple yet mechanistically versatile antiviral metabolites.