The S-Adenosylmethionine (AdoMet) decarboxylase inhibitor 5'{[(Z) 4- amino-2-butenyl]methyamino}-5-deoxyadenosine (MDL73811), cures drug refractory T. b. rhodesiense clinical isolates in model infections; and is actively taken up via a purine transporter. We will study the mode of action of these agents and identify other potential drug candidates. Both MDL73811 and DFMO dramatically elevate AdoMet due to an unregulated AdoMet synthetase. AdoMet is aminopropyl group donor for spermidine synthesis and methyl group donor for most methyltransferases. We believe the extremely high (5mM) AdoMet pools in treated parasites (not in host cells) leads to hypermethylation of cell components. The flow of [35S]methionine through AdoMet, adenosylhomocysteine (AdoHcy) and increased incorporation of U[14C] as opposed to [35S]methylthioadenosine. We will study: 1. The ability of arsenical drug resistant clones of T. b. rhodesiense to transport purine nucleoside-based analogs of AdoMet, AdoHcy and methylthioadenosine, 2. Partitioning of methionine cycle intermediates by use of polyamine, transmethylation and transsulfuration inhibitors, 3. Examine protein and phospholipid methylation in normal and drug treated cells, and determine whether elevated AdoMet levels during treatment result in hypermethylation, 4. Transmethylation during transformation of long slender (LS) bloodforms to procyclic forms, we will monitor the release of variant surface glycopeptide from LS forms and the development of procyclin a surface protein of procyclic forms, and 5. DFMO refractory T. b. rhodesiense isolates for altered polyamine or transmethylation patterns, This work will utilize bloodforms grown in culture; HPLC and radiodetection to study the fate of AdoMet and methionine and isoelectric focusing to identify methylated proteins. We hope to determine the roles(s) of transmethylases and their control in African trypanosomes and their importance in the life cycle.
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