Polyamines, low molecular weight compounds common to all cells, are essential to growth and differentiation, acting as cofactors in macromolecular synthesis and stabilizing nucleic acids. This study aims to a) explore regulation of synthesis of polyamines and related nucleosides in pathogenic African trypanosomes; b) ascertain functions of polyamines in these parasites; c) develop novel single drug and combination chemotherapy incorporating polyamine antagonists; d) correlate changes in polyamine synthesis to life-cycle stages of Trypanosoma b. brucei. The research will exploit our preliminary findings in which a specific polyamine antagonist, DL-Alpha-difluoromethylornithine (DFMO) cured a short-term murine infection, and DEMO + the antitumor agent bleomycin cured a long term CNS infection. A key enzyme of polyamine metabolism is ornithine decarboxylase (ODC), the target of DFMO. Since ODC activity is carefully regulated in most cells, the study will examine ODC induction as it relates to bloodstream/culture trypomostigote transformation by charting ODC changes in bloodstream forms on subculture, and also in a lower trypanosomatid. A polyamine-activated protein kinase, phosphorylating an endogenous substrater in T. b. brucei will be examined as ODC regulator. A byproduct of polyamine synthesis, methylthioadenosine (MTA), may be crucial for recycling adenine and methionine. A key enzyme in this pathway, MTA phosphorylase will be studied in T. b. brucei, as will novel inhibitors of the enzyme, and intermediates in MTA breakdown. Trypanosomatid DNA plymerases are polyamine-activated. Their interaction with kDNA as template primers, and effects of cationic trypanocides which mimic polyamines, will be examined. To guide combination chemotherapy, effects of polyamine depletion of the the parasite on uptake of polyamine-like antitumor and other agents will be determined. These studies will correlate in vivo, in vitro (whole cell), crude and purified enzyme studies, and employ radio-tracer techniques as well as thin-layer and high-precision liquid chromatography. Ion-exchange and affinity chromatography would be used in ODC, protein kinase and DNA polymerase studies. We would further investigate polyamine-cationic drug interaction in vivo by combination therapy with cationic antitumor agents + polyamine antagonists. We hope thereby to uncover additional targets for chemotherapy and to develop novel agents or combinations of agents useful for human and veterinary trypanosomiases.
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