Isolation of Actinomycin D from marine fauna associated bacteria

Two antibiotic producing bacterial strains, one (SS23) from marine sediments and another (CN3) from the venom duct of the marine cone snail Conus monile have been isolated. On the basis of morphology, phenotypic and genotypic characteristics, the bacteria have been shown to belong to the genus Streptomyces. Purification of bioactive principles was achieved by solvent extraction, fractionation by silica gel chromatography and finally by high performance reverse phase liquid chromatography using a C18 column. Antimicrobial activity studies were carried out to identify bioactive fractions. Of the HPLC fractions, one fraction a MIC of 1 mg/ml against B. subtilis, E. coli and methicillin resistant S. aureus and a MIC of 50 mg/ml against C. albicans. Mass spectrometric analysis showed that this HPLC fraction had a single compound that had a mass of 1255 Da. Further activity studies showed that this molecule exhibited potent activity against M. tuberculosis (MIC of 10 mg/ml). The compound also showed potent cytotoxic activity against breast (MCF-7), melanoma (A375), prostate (DU145) and lung (A549) cell lines at IC concentration values of 20 mg/ml. Molecular structural analysis of this compound using a combination of tandem mass spectrometry and multidimensional NMR spectroscopy (Figure.1) has shown that this compound is Actinomycin D. Sequencing of the genomic DNA of SS23 and CN3 are currently in progress to establish the identity of the species.

Fig. 1: Finger print region (H^N-Hα) of the two dimensional ¹H-¹H TOCSY NMR spectrum. Residues specific assignments are indicated in the spectrum.