ABSTRACT
Chuangxinmycin (CM) from Actinoplanes tsinanensis was an antibiotic discovered by Chinese scientists about 40 years ago. It contains a new heterocyclic system of indole fused with dihydrothiopyran, whose biosynthetic mechanism remains unclear. CM is used as an oral medicine in the treatment of bacterial infections in China. The simple structure makes CM as an attractive candidate of structure modification for improvement of antibacterial activity. Recently, we analyzed the secondary metabolites of Actinoplanes tsinanensis CPCC 200056, a CM producing strain, as a natural CM analogue. We discovered the first natural CM analogue 3-demethylchuangxinmycin (DCM) as a new natural product. Compared to CM, DCM exhibited a much weaker activity in the inhibition of the bacterial strains tested. The finding provides valuable information for the structure-activity relationship in the biosynthesis of CM.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Micromonosporaceae/chemistry , Anti-Bacterial Agents/chemistry , China , Indoles/chemistry , Indoles/isolation & purification , Structure-Activity RelationshipABSTRACT
PURPOSE: To find novel potential and less toxic benquinone anamycin heat shock protein 90 (Hsp90) inhibitors as anticancer agents, a limited series of 17-substituted or 17,19-disubstituted 17-demethoxygeldanamycin analogs were synthesized and tested for anti-proliferation activity against human cancer cells. Liver toxicity was also tested in vivo. METHODS: Five 17-alkylamino-17-demethoxygeldanamycins and two 17-alkylamino-19-methylthio-17-demethoxygeldanamycins were synthesized from geldanamycin (GA) and 19-methylthiogeldanamycin (19-S(methyl)-GA), respectively. Anti-proliferation activities of the GA analogs were determined in MCF7, HeLa, HCT116 and HepG2 cells using the MTT method. Western blot and cell cycle analyses were performed for mechanistic study. The growth inhibition effect of potential geldanamycins was also investigated in normal Buffalo rat liver (BRL) cells. In vivo liver toxicity was tested in Institute of Cancer Research (ICR) mice by tail vein injection of the tested compounds. RESULTS: Most of the 17-alkylaminogeldanamycins exhibited obvious growth inhibition effects on multiple human cancer cell lines. The anti-proliferation activity of 19-methylthio-substituted geldanamycins was significantly lower compared with no 19-substitution geldanamycins in all tested cancer cells. The compound 1b (17-[2-(piperidinyl-1'-yl)-ethylamino]-17-demethoxygeldanamycin) exhibited the highest anti-proliferation activity in MCF7, HeLa and HCT116 cells, which was much more effective than GA and the developing Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin). Meanwhile, compound 1b exhibited weaker growth inhibition effect on BRL cell line than GA and 17-AAG. 1b induced cell cycle arrest at the G2/M phase in MCF7 cells. Cleavage of PARP associated with apoptosis and degradation of the Hsp90 client protein Akt and Her2 was also induced by treatment of 1b in HeLa and MCF7 cell lines. In spite of the relatively weaker activity of 1b compared with GA and 17-AAG against HepG2 cells, 1b was further identified with lower hepatotoxicity than GA in vivo. CONCLUSION: Compound 1b is regarded as a new potential Hsp90 inhibitor with low hepatotoxicity for further study.
Subject(s)
Antibiotics, Antineoplastic/chemical synthesis , Antibiotics, Antineoplastic/pharmacology , Benzoquinones/chemical synthesis , Benzoquinones/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Lactams, Macrocyclic/chemical synthesis , Lactams, Macrocyclic/pharmacology , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/toxicity , Benzoquinones/chemistry , Benzoquinones/toxicity , Cell Culture Techniques , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathology , Female , HCT116 Cells , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HeLa Cells , Hep G2 Cells , Humans , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/toxicity , Liver/cytology , Liver/drug effects , Liver/pathology , Liver Function Tests , MCF-7 Cells , Male , Mice , Mice, Inbred ICR , Molecular Structure , Rats , Structure-Activity RelationshipABSTRACT
Microbial secondary metabolites are one of the major sources of anti-bacterial, anti-fungal, antitumor, anti-virus and immunosuppressive agents for clinical use. Present challenges in microbial pharmaceutical development are the discovery of novel secondary metabolites with significant biological activities, improving the fermentation titers of industrial microbial strains, and production of natural product drugs by re-establishing their biosynthetic pathways in suitable microbial hosts. Synthetic biology, which is developed from systematic biology and metabolic engineering, provides a significant driving force for microbial pharmaceutical development. The review describes the major applications of synthetic biology in novel microbial secondary metabolite discovery, improved production of known secondary metabolites and the production of some natural drugs in genetically modified or reconstructed model microorganisms.
Subject(s)
Bacteria/metabolism , Biological Products/metabolism , Drug Discovery , Fungi/metabolism , Synthetic Biology , Fermentation , Gene Transfer Techniques , Metabolic EngineeringABSTRACT
Spiramycin (SP) belongs to the 16-member macrolide antibiotics. It contains three components,namely SP I, SP II and SP III, which differ structurally in the acylation moieties on the C3 of the lactone. The SP I component contains a hydroxyl group at C3. SP II, and SP III are formed by further acetylation or propionylation of the C3 of SP I, by the same 3-O-acyltransferase (3-O-AT) . The study focused on simplifying spiramycin components. Theoretically, disruption/deletion of the 3-O-AT gene will reduce/stop the acylation of SP I to SP II and SP III. In this study, degenerated primers were designed according to the conserved regions of 3-O-acyltransferase, MdmB and AcyA in the medicamycin and carbomycin producers of S. mycarofaciens and S. thermotolerans, respectively, and an 878bp DNA fragment was amplified from the spiramycin-producer of S. spiramyceticus F21. Blast analysis of the 878bp DNA fragment suggested that it encoded the 3-O-acyltransferase (3-0-AT, sspA) gene for spiramycin biosynthesis. The flanking regions of this 878bp DNA fragment were then amplified by single-oligonucleotide-nested PCR, and a total of 4.3 kb DNA was obtained (3457nt among the 4.3kb fragment was sequenced, and deposited in GenBank DQ642742),covering the whole putative 3-O-acyltransferase gene, sspA. The sspA was then deleted from the S. spiramyceticus F21 genome by double cross-over homologous recombination, mediated by temperature-sensitive plasmid pKC1139. A comparison was done of the components of spiramycins produced by the sspA-deleted mutant strain with that of the parent strain by HPLC analysis, which showed that sspA-deleted mutant produced SP I (72%), SP II (18%), and SP III (9.6%), whereas parent strain produced SP I (7.8%), SP II (67%), and SP III (25%), respectively, demonstrating the role of ssp A in the acylation of SP I into SP II and SP III. The ssp A-deleted mutant strain obtained in this study may be used for the production of SP I, or may serve as a good starter for the construction of spiramycin derivatives.
