A natural depsipeptide antibiotic binds the E-site of the bacterial ribosome

Key Points:

• Researchers screened a WAC6 pre-fractionation library against a hyperpermeable efflux-deficient E. coli strain to identify antimicrobial activity, using automated liquid handling and optical density measurements.
• The study involved purification and structural characterization of novel compounds, MKM-A and MKM-B, from Streptomyces rimosus cultures, employing chromatography, mass spectrometry, NMR, and amino acid stereochemistry analysis.
• Whole-genome sequencing and biosynthetic gene cluster (BGC) analysis identified the MKM biosynthetic genes, which were cloned and heterologously expressed in Streptomyces coelicolor for functional studies.
• Antimicrobial efficacy of MKMs was assessed via MIC determination against various bacteria, cytotoxicity testing on mammalian cells, hemolysis assays, time-dependent killing, and membrane permeability assays; resistance development was studied through serial passaging and mutation analysis.
• Advanced structural studies including cryo-electron microscopy of MKM–ribosome complexes, toeprinting, and in vitro translation assays elucidated the mechanism of action, while pharmacokinetic and in vivo assays in mice and C. elegans demonstrated MKM-A’s stability and therapeutic potential.