Microbial Source Of Human Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase (kdr-cd) Inhibitors

Angiogenesis, the process by which new blood vessels developed from pre-existing blood vessels and vascular endothelial cells, is important in many disease states including inflammation, cancer, wound healing, ischemic cardiovascular disease, psoriasis, peptic ulcers, rheumatoid arthritis, atherosclerosis, macular degeneration. In new growth, the growth factors promote angiogenesis, which includes normal growth patterns and progression such as cell division, migration until vessels can function. Firstly, Virchew and Thiersch identified the presence of a capillary network in tumors and supposed its generation from the host vascular bed in 1863. After one hundred years, Folkman proposed that tumor blood vessel formation was dependent on a tumor angiogenic factor (TAF) and targeting the tumor's dependence on angiogenesis could effectively treat cancer. The fact that several new drugs such as bevacizumab, Sunitinib and sorafenib, target specific molecules involved in angiogenesis validate Folkman's hypothesis that tumor growth can be inhibited by attacking the vasculature with specific angiogenesis inhibitors.Vascular endothelial growth factor (VEGF) acts as a highly specific mitogen directly involving in the process of angiogenesis, which induce endothelial cells division and proliferation and also improves vascular permeability. As a potent multifunctional cytokine, the highly conserved glycoprotein VEGF plays an important role on the vascular endothelium and is probably critical to new tumor vascular formation. VEGF helps maintain the viability of immature vasculature and derives expression of the antiapoptotic proteins Bcl-2 and Al, which enhances cell survival. The VEGF protein also regulates hematopoietic stem cell survival and induces angiogenesis via a direct effect on endothelial cells by binding to KDR. Moreover, VEGF is the most selective vascular endothelial cell mitogen known. Scientists have identified elevated VEGF levels in many tumors. They have also found that overexpression of VEGF correlates with increased risk of metastatic disease and overall poor prognosis in different cancers.VEGF performs its function via binding to special receptors; so far, three high-affinity cognate endothelial receptors for VEGF have been identified: VEGFR1/Fltl, VEGFR2/Flkl/KDR and VEGFR3/Flt4. Studies have demonstrated that Flk-1/KDR undergos strong ligand-dependent tyrosine phosphorylation in intact cells and mediated mitogenesis and chemotaxis in response to VEGF, while Flt-1 reveals weak or undetectable responses. So that KDR is the major signal transducer for the division and proliferation of endothelial cells. VEGF binding to KDR results in dimerization of receptor monomers, transphosphorylation by dimerized receptors and docking of signaling proteins to receptor phosphotyrosines. KDR undergos dimerization and autophosphorylation, thus initiating a cascade of downstream signaling events, resulting in endothelial cells-associated reaction. Tumors require nutrients and oxygen in order to grow, and new blood vessels, formed by the process of angiogenesis, provide these substrates. As angiogenesis is of crucial importance for tumor growth, Therefore, KDR is an appealing target for anticancer therapeutics. Effectively inhibiting VEGFR2 signaling is very attractive for scientists to design and screen new anticancer agents against tumor angiogenesis.With the recombinant KDR-CD as target, an ELISA assay for screening new antagonists of KDR-CD was developed by us. And the secondary metabolites produced by three streptomyces strains named as I06A-02754, I06A-02832 and I06A-03304 separatively were identified with activity against KDR-CD in this assay. Separated by several chromatography methods, ten compounds,2754A, 2754B,2754C,2754R,2832A,2832B,2832C,3304A,3304B and 3304C were separated and purified from the cultured broth of Streptomycete 2754,2832 and 3304. The structure of them were elucidated by UV, IR, MS,1H-NMR, 13C-NMR, DEPT, 'H-'H COSY, HMQC and HMBC.2754B,2754C,2832A,2832B,2832C,3304A,3304B and 3304C were identified as nanaomycin a A, nanaomycinβA, juglomycin D, Trp, cyclo-(Tyr-Pro), cyclo-(Phe-Gly), cyclo-(Leu-Pro), cyclo-(4-hydroxyl-Pro-Phe) and cyclo-(Pro-Phe) separatively.2754A was a novel compound. The ten compounds were screened for their KDR-CD antagonist effects.2754A has no inhibitory activity against methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Bacille Calmette Guerin, Pasteurella, Aeruginosus Bacillus, Escherichia coli, Candida albicans and H37Rv(CSU) at 100μg ml-1 and also has no any cytotoxicities on the HCT-8 (human coloncancer), Bel-7402 (human hepatocarcinoma) and BGC-823 (human gastric carcinoma) cell lines at 10μmol 1-1 by the MTT assays. All the ten compounds were the first research report about their KDR-CD antagonist activities.