Polyamines are low molecular weight amines, necessary for growth and differentiation of cells. We established that an inhibitor of polyamine biosynthesis DL-alpha-difluoromethyl-ornithine (DFMO), cures model infections of African trypanosomes (Trypanosoma brucei ssp). Clinical trials (>200 cases) in West Africa indicate that DFMO is >90% effective against CNS disease refractory to Melarsoprol. Despite these advances these are concerns associated with further development of DFMO: a) early trials in East Africa indicate that DFMO refractory strains of T. b. rhodesiense occur, b) prolonged DFMO treatment is required, and c) resistant strains will probably emerge in West Africa. For these reasons and based upon the trypanocidal action of DFMO, we intend to pursue polyamine metabolism in African trypanosomes to: a) Examine the basis for natural resistance to DFMO in T. b. rhodesiense; b) develop alternative chemotherapy against polyamine biosynthesis and related pathways using novel polyamine analogs and other inhibitors; c) study the metabolism of the key intermediate, S-adenosylmethionine (SAM) which is essential for both polyamine formation and methylation; d) determine if SAM overproduction affects methylation of macromolecules and is centrally involved in transformation of bloodstream trypomastigotes. The unexpected and dramatic rise in the methylation index (SAM: S- adenosylhomo-cysteine) and 5x increase in Protein Methylase II activity suggests that SAM synthetase (which forms SAM from methionine and ATP) is deregulated upon exposure to DFMO, potentially leading to hypermethylation. We will study: 1) SAM synthetase activity in naturally transforming cells and those induced to transform by DFMO; ii) activities of protein and nucleic acid methylases; iii) components within the cell which become methylated during transformation. In addition, we will examine inhibitors of SAM synthetase and transmethylation in an effort to determine their regulatory role during development, and as potential targets for chemotherapy. To further develop treatment of T. b. rhodesiense we will study DFMO refractory isolates to determine possible mechanisms of resistance, including the overproduction of polyamines or ODC, alterations in trypanothione levels, changes in uptake or efflux of DFMO, and possible amplification of the ODC gene.
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