The major goal of this proposal is to develop and test """"""""second generation inhibitors of polyamine biosynthesis, most notably compounds that inhibit adenosylmethionine decarboxylase. Although methylglyoxal bis(guanylhydrazone) (MGBG) has extremely narrow therapeutic index and the compound exerts effects, which in all likelihood are unrelated to the biosynthesis of the natural polyamines, it still remains the compound of choice, when the synthesis of new inhibitors ar designed. In the MGBG molecule two very important properties are combined: it is extremely potent competitive inhibitor of adenosylmethionine decarboxylase, an enzyme needed for the synthesis of spermidine and spermine, and it utilizes the polyamine-deprivation inducible transport system. Thanks to our very recent syntheses of new MGBG analogs, we have got a much better insight into the functional structure of MGBG molecule as regards the inhibition of adenosylmethionine decarboxylase, the use of the native polyamine carrier for cellular transport, the inhibition of diamine oxidase and the capabiity of replacing intracellularly bound polyamines. We have already synthesized several analogs of MGBG, designed synthetic routes for many further compounds and started to test a few compounds in combination with difluoromethylornithine (DFMO) in animal tumor screens. We have also designed experimental approaches to evaluate the significance in vivo of the diamine oxidase inhibition produced by the diguanidines. Furthermore, based on historical evidence, both experimental and clinical, and supported by our preliminary experiments, we have uncovered a possible biochemical basis for some major side-effects of MGBG, such as hypoglycemia and disturbances affecting muscle tissue. These side-effects could conceivably be based upon an inhibition of carnitine-dependent fatty acid oxidation by bis(guanylhydrazones). Finally, using hybridization techniques (with the aid of a pBR322 plasmid containing an ornithine decarboxylase gene insertion) we will elucidate the nature of cellular compensatory mechanisms triggered by polyamine deprivation at the level of gene expression. With the aid of sandwich hybridization method we developed for ornithine decarboxylase, we have shown that in tumor cells growing under the pressure of DFMO, the expression of the enzyme is greatly enhanced.
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