Study on biologically active compounds isolated from streptomyces and echinomycin derivatives

Abstract

The current study is undertaken to investigate the new tumor cell growth inhibitors derived from Streptomyces. The producing organism was isolated from soil collected at the Wonju area (CHIAK mountain) in Kangwon-Do, Korea. The 800 microorganisms were tested in primary screening. Primary screening tests were performed using antimicrobial activity and tumor cell cytotoxicity in vitro. Cultured broth of microorganism was tested for antimicrobial activity against Staphylococcus aureus and for cytotoxicity against P 388 murin cell line. Finally, we chose the SK-302 which has a excellent antimicrobial activity and tumor cell cytotoxicity. The selected microorganism SK-302 belongs to Actinomycetes Streptomyces sp. based on morphological and cultural properties on various medium. The SK-302 was cultivated in large scale, lab and 300L pilot-scale fermentor, to prepare the sample. Active metabolites were isolateded from cultured broth by several isolation steps of extractions and column chromatography (silica column). Purification was carried out using the Prep-HPLC with ODS column and 1H-2H polystylene gel column. As a result, about 500 mg of pure SK-302B and 27 mg of pure SK-302C were obtained from cultured broth. Pure SK-302B and SK-302C were tested in vitro (3 LL, B 16 mouse tumor cell), in vivo (P 388, B 16, S-180 mouse tumor cell), in vitro (SK-MEL-2, HCT-15, SNU-1, KATO human tumor cell) tests, based on chemosensitivity tests (cytotoxicity and colony forming inhibition assay). Structural study of purified fractions were carried out several spectroscopic methods (2D FT-NMR and APCI, FAB MASS) and amino acid analysis. As a result, SK-302B (mol.wt 1101.3) is a cyclooctapeptide (essential amino acid: Ala, Ser, non-essential amino acid N-Me Cys, N-Me Val) with aromatic (quinoxaline) moiety. The candidate compound for antitumor agent, SK-302B, was finally identified as a known compound, echinomycin A (quinomycin A). Echinomycin and It``s synthetic analogue TANDEM i...