Mithramycin (MTM) is an aureolic acid antimicrobial and antitumor agent produced by various Streptomyces species including S. argillaceus, which has been used, e.g., for the treatment of testicular carcinoma. In addition, MTM is unique among anticancer agents in that it also has been used clinically to treat cancer-caused malignant hypercalcemia and Paget's bone disease. However, MTM's bone marrow, hepatic, and renal toxicity limit its widespread clinical use. It is proposed to investigate various aspects of the biosynthesis of the antitumor and osteoclast-inhibiting agent mithramycin in order to develop analogs with increased therapeutic indices, which also may allow the separation of the two principal effects of MTM, (i) on cancer growth and (ii) on osteoclasts. This will lead to novel antitumor agents and/or to therapeutics against osteoporosis and other diseases related to bone growth disorders, and bears the potential for a novel gene therapy concept in future. Combinatorial biosynthetic methods will be used to provide an array of MTM analogs. For this, the biosynthetic pathway to MTM, which is dominated by a type II polyketide synthase (PKS), needs to be further characterized. Especially genes encoding enzymes responsible for the late biosynthetic steps, the post-PKS tailoring enzymes, particularly oxidoreductases and group transferases will be modified Resulting mutant strains will help to determine the series of events within the biosynthetic pathway and will characterize substrates and function of important enzymes in the MTM pathway. This information will be used to design novel compounds with specific activity-increasing functionality. In context with the mechanism of action of MTM on osteoclasts, we want to explore whether MTM derivatives can effect the expression of c-src, a proto-oncogene necessary for the osteoclastic bone resorption, following the novel hypothesis that MTM and its derivatives inhibit osteoclast bone resorption by blocking Sp 1 binding to the promoter region of the c-src proto-oncogene. The following three specific aims will be addressed: (1) To further characterize the biosynthetic pathway of mithramycin and to develop new niithramycin derivatives through selective gene inactivation and product identification. Various group transferases and oxidoreductases will be investigated. In addition, the mtm genes will be recombined with promising deoxysugar biosynthesis, glycosyltransferase and oxygenase encoding genes from other pathways to develop novel niithramycin analogs modified in their saccharide and/or 0-atom pattern. (2) The two oxygenases of the MTM pathway, MtmOII and MtmOIV, will be investigated. The work on MtmOII, an early-acting oxygenase, will help to identify the missing link between the final PKS product and 4-demethylpremithramycinone, the earliest mithramycin precursor documented to date. Tetracyclic niithramycin analogs will be converted into their tricycic and expected more active counterparts by overexpressing oxygenase MtmOIV in the various glycosyltransferase deletion mutants. (3) To assay the binding properties of MTM and its novel analogs to the GC-rich elements in the c-myc and c-src promoters and their ability to prevent Sp 1 binding. Testing the high-affinity c-myc will follow thisand c-src binding compounds for inhibition of gene expression in human cancer cells and for their effects on growth and viability of normal and cancer cells. Finally, promising c-src inhibiting analogs will be analyzed for activity against osteoclastmediated bone resorption.
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