Human African trypanosomiasis (HAT) is caused by the parasitic protozoan, Trypanosoma brucei. HAT is listed as a Category 1 diseases (emerging and uncontrolled) by the WHO and it exerts a large burden in both health and economic costs to the affected regions. There is a great need to translate recent advances in the understanding of the basic biology of the parasite into new drugs. Polyamines are essential metabolites that are required for cell growth. The polyamine biosynthetic enzymes, including ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) are essential to the parasite and have been the target of anti-proliferative chemotherapy. The most successful example is the treatment of HAT by a-difluoromethyornithine (DFMO), a suicide inhibitor of ODC. In the trypanosomatid parasites the polyamine metabolic pathway is unique. Spermidine is conjugated to glutathione to produce an unusual cofactor termed trypanothione, required for maintenance of reduced thiol pools and trypanosomatids lack catabolic enzymes found in the host. Further the mechanisms for regulating the pathway differ from the host. We recently discovered that AdoMetDC is allosterically activated by formation of a heterodimer with an inactive paralog (prozyme), which is present only in the trypanosomatid parasite. Furthermore we have found evidence that translational regulation of prozyme levels may be a major factor in controlling AdoMetDC activity, and thus polyamine and trypanothione levels in the parasite. We plan a set of comprehensive studies to address both the biology of polyamine and trypanothione metabolism in T. brucei, and to exploit our findings for the identification of potential lead compounds for the development of new anti-trypanosomal agents. The studies will build on our existing data to address several important questions.
In Aim one we will continue our studies of transgenic parasites to identify and characterize regulatory mechanisms that control the expression levels of the pathway enzymes, and in turn regulate metabolic flux. Study of pathway regulation is important to identify the enzymes that serve as the control points in polyamine biosynthesis, and thus are likely to be the best targets for drug discovery projects. Furthermore the discovery of additional unique regulatory mechanisms may lead to new approaches to influence the pathway function, and thus parasite growth.
In Aim two we will conduct structural studies to elucidate the molecular details of the activation of AdoMetDC by prozyme. These studies will inform drug discovery efforts to identify and optimize inhibitors of AdoMetDC and they will provide insight into the general question of the molecular basis for allosteric regulation of protein function.
In Aim 3 we will conduct a high through put screening (HTS) campaign to identify new inhibitors of both ODC and AdoMetDC, and we expect to identify inhibitor series against both enzymes with anti-trypanosomal activity that have the potential to form the basis of future lead optimization programs to develop new agents to combat HAT.
Human African sleeping sickness is a fatal insect borne disease that if untreated leads to death, yet current drug therapies are toxic and difficult to administer. The work described in this proposal characterizes the biology of an essential metabolic pathway (polyamine and trypanothione biosynthesis) that has unique features in the parasite, with the goal of developing new chemotherapeutic agents against the disease.
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