This is an investigation of purine and purine analogue metabolism in procyclic and bloodstream forms of the African trypanosomes. Whole cell metabolic studies will involve incubation with radiolabeled purine or purine analogues followed by acid extraction and analysis by high performance liquid chromatography and liquid scintillation counting. Prior studies have indicated that C nucleoside analogues of insoine, such as Formycin A and 9-deazainosine, are very effective against these parasites. Using the above techniques along with computer analysis of metabolic flow, it is hoped that the particular enzymes involved with the activation of the purine analogues and/or those that are inhibited by them or their metabolites will be identified. Using standard enzyme purification techniques those enzymes identified by these metabolic studies will be investigated as to their kinetics and substrate and inhibitor specificities. These results will then be compared to those of equivalent mammalian enzymes. The last phase of this proposal will involve the elucidation of the mechanism of action of purine analogues such as Formycin B and 9-deazainosine on the African trypanosomes. Using standard cell biology techniques, two possibilities will initially be studied: the inhibitory role of the high levels of IMP analogues formed from these compounds on the organism's ability to salvage purines, and the effects of the incorporation of their respective ATP analogues into these parasites nucleic acids. It is hoped that the results of these investigations will result in the identification of metabolic differences between the African trypanosomes and their mammalian hosts that can be exploited in the development of chemotherapeutic agents.
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