The Discovery And Identification Of New Herbimycin Analogues And Other Secondary Metabolites From Streptomyces

Streptomyces are well known for their potential to produce secondary metabolites with various chemical structures and biological activities, and play an important role in discovery and development of new microbial drugs. Chemical screening is a powerful method for the systematic study of secondary metabolites produced by microorganisms.This dissertation is divided into two parts. The first part describes the identification of two novel natural herbimycin analogues from Streptomyces sp. CPCC 200291. The second part involves the exploration and identification of quinomycins and other secondary metabolites from strains of the genus Streptomyces.Herbimycin A (HBM A) is a benzoquinone ansamycin and shares the similar structure with geldanamycin. Herbimycin A possesses strong cytotoxicity against tumor cells, as it is a specific inhibitor of heat-shock protein 90, whose client proteins have important roles in cell-cycle regulation and acts as a lead compound in development of anticancer drugs. New analogues of herbimycin A with higher water solubility and lower hepatoxicity may provide candidates in anticancer drugs.Herbimycin A undergoes a very complex post-PKS tailoring process, which involves carbomoylation, oxidation, hydroxylation, O-methylation, in its biosynthesis. This process may generate some herbimycin analogues resulted from incomplete post-PKS tailoring modifications of progeldanamycin, the precursor compound formed by the catalyzation of type I polyketide synthases (type I PKS). These natural /biosynthetic herbimycin analogues may provide good candidates in herbimycin development.Streptomyces sp. CPCC 200291, a soil isolate of Hainan Province, China with strong antibacterial and antifungal activities, is preserved in China Pharmaceutical Culture Collection (CPCC). In the course of studying the secondary metabolites of the isolate, which is grown by solid-state fermentation, we identified herbimycins A and C as the principal components by TLC, HPLC and LC-MS, and discovered two new herbimycin analogues as minor components,compound 1 and 2. The structure of the two analogues were determined to be 4,5-dihydro-(4S)-4-hydroxyherbimycin B (named herbimycin G, HBM G) and (15R)-15-hydroxyherbimycin B (named herbimycin H, HBM H) by extensive spectroscopic data analysis, including HRESIMS,2D NMR、 Mosher’s reaction. Compound 1 is probably a shunt metabolite in the biosynthesis pathway to herbimycin A without the benefit of the normal post-PKS tailoring enzymes, while compound 2 is presumably an intermediate of herbimycin A biosynthesis. Both compound 1 and 2 displayed decreased cytotoxicity against HCT116、 Hela、A549 and HepG2 cells but much increased water solubility.As mentioned above, Streptomyces produces abundant secondary metabolites with various biological activities. We made an initial inspection of the secondary metabolites from nearly 30 strains of Streptomyces provided by CPCC, looking for secondary metabolites with potential as drug candidates.The secondary part of this dissertation describes the identification of quinomycins from Streptomyces sp. CPCC 200497 and doxorubicin derivatives from Streptomyces sp. CPCC 204110 by chemical screening methods such as TLC, HPLC and LC-MS. In addition, a preliminary comparison of the quinomycin(s)/echinomycin produced by three known quinomycin producing strains, CPCC 200239, CPCC 200240 and CPCC 200299, with that of CPCC 200497 was conducted.Streptomyces sp. CPCC 200497 is a soil isolate with antivirus activities collected from Guangxi Province, China. The EtOAc extracts of the culture of this strain was fractionated by preparative TLC and then analyzed by HPLC-UV-DAD, and ESI(+/-)MS, to obtain data such as UV-Vis, molecular mass and formula, etc. Quinomycin A and C were identified as two principal components, and quinomycin B as one minor component. Quinomycin A and C showed remarkable inhibition activities against Gram-positive bacterial strains including MRSA and VRE. For example, the MICs of quinomycin A and C against ATCC 33591 (Staphylococcus aureus, MRSA) are 0.06 and≤0.015, respectively. The MICs of quinomycin A and C against ATCC 51299(Enterococcus faecalis, VRE) are both≤0.015, even lower than vancomycin and clarithromycin.As a depsipeptide antibiotics, quinomycin contains a pair of quinoxaline or quinoline chromophores in its structure. It shows antibacterial, antiviral and antitumor cell activities because the chromophores of the molecule can bind strongly to the double helix of DNA, which interferes replication and transcription. In 1980s, as an antitumor drug candicate, quinomycin A entered a phase II clinical trial, but no significant anti-tumor effects were observed.The identification of CPCC 200497 as a quinomycin producer and the very strong antibacterial activities of quinomycins against Gram-positive bacteria strains, suggest that quinomycins may be promising candidates for antibacterial drug development. CPCC 200497 is a good producer for preparing quinomycins.CPCC 200497 can also produce other secondary metabolites with different structures from quinomycins, as we have detected some other information of compounds, and deserved for further study.Streptomyces sp. CPCC 200239 and CPCC 200240 produce quinomycin A only, while CPCC 200299 produces both quinomycin A and C. Besides, CPCC 200240 is found to produce enopeptin A, a depsipeptide compound with anti-Gram positive bacterial activites.Streptomyces sp. CPCC 204110 (I10A-00954) is a soil isolate from Qinghai Province, China. It may be a producer of doxorubicin (or adriamycin) based on the datas of HPLC-UV-Vis-MS of the EtOAc extracts of this strain. Doxorubicin may be acetylated in the EtOAc extracts. Doxorubicin belongs to the anthracycline antibiotics. It is a non-specific inhibitor of eukaryotic cell cycle, and plays an important role in cancer chemotherapy.