The long-term goal of the proposed research is to investigate whether the enzyme oleate desaturase (or delta-12 desaturase) of Trypanosoma cruzi (ODTc) can be used as a target for developing novel chemotherapeutic agents against this pathogen. T. cruzi, the etiologic agent of Chagas' disease, expresses ODTc that is essential for the conversion of oleic acid (C18:1) into linoleic acid (C18:2) by a dehydrogenase reaction. Linoleic acid and its downstream product linolenic acid (C18:3) are critical for maintaining fluidity and the architecture of T. cruzi plasma membrane during temperature variation throughout the parasite life cycle. Interestingly, ODTc is unique in a sense that this enzyme, or its putative homologue, is not present in humans. Therefore, it could serve as an excellent molecular target for therapeutic purpose against T. cruzi infection. To validate ODTc as target, we aim to study the essential nature of this enzyme by biochemical and molecular methodologies. In this regard, two strategies will be pursued.
In Aim -1, an attempt will be made to silence the expression of ODTc gene using two approaches. First, we will perform the knockout experiment. Second, the expression of ODTc will be down-regulated using antisense phosphorothioate oligonucleotides. The test of viability and infectivity, as well as the investigation of the biochemical properties of ODTc null mutant will provide important information regarding the role of this enzyme in T. cruzi.
In Aim -2, the development and testing of ODTc inhibitors will be carried out in vivo and in vitro. We propose to generate selective inhibitors for ODTc by modifying the structure of known inhibitors for delta-6 and delta-9 desaturases. A high-throughput screening of the drugs will be performed in epimastigote and trypomastigote forms using a modified viability (MTT) assay. The next step will be to confirm the specific inhibition of ODTc by metabolic labeling. Finally, the inhibitors will be tested in vivo using the murine model of Chagas' disease. Side by side, we will also test the efficacy of different antisense oligonucleotides in vivo. If successful, the proposed study will validate our hypothesis that ODTc is one of the best targets for designing new drugs against T. cruzi. Currently, there is only one commercial drug available for the treatment of Chagas' disease. The appearance of T. cruzi resistant strains clearly points out the urgent need for new medications against this infectious disease, which affects millions of people in Latin America, and it is an emergent public health concern in the United States.
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