Oleate Desaturase: Novel Drug Target for Chagas Disease

这项拟议研究的长期目标是调查油酸去饱和酶(或 克氏锥虫(ODTC)的Delta-12去饱和酶可作为开发新的靶点 针对这种病原体的化疗药物。查加斯病的病原体克氏锥虫表达了 脱氢酶将油酸(C18：1)转化为亚油酸(C18：2)所必需的ODTC 反应。亚油酸及其下游产物亚麻酸(C18：3)是维持流动性的关键 克氏毛滴虫的质膜结构在整个寄生虫生命周期中的温度变化。 周而复始。有趣的是，ODTC是独一无二的，因为这种酶或其假定的同系物不存在于 人类。因此，它可以作为治疗克氏毛滴虫的一个很好的分子靶点。 感染。为了验证ODTC作为靶点，我们旨在通过生化方法研究该酶的基本性质 和分子方法论。在这方面，将采取两项战略。在AIM-1中，将尝试 用两种方法沉默ODTC基因的表达。首先，我们将进行淘汰赛 做实验。其次，使用反义硫代磷酸将下调ODTC的表达 寡核苷酸。活力性、感染性试验及生化特性调查 这将为该酶在克氏锥虫中的作用提供重要的信息。在AIM-2中， ODTC抑制剂的开发和测试将在体内和体外进行。我们建议 通过修饰已知的Delta-6和Delta-9抑制剂的结构来产生ODTC的选择性抑制剂 降低饱和度。药物的高通量筛选将在上鞭毛体和类鞭毛体中进行。 采用改良的存活率(MTT法)分析。下一步将确认ODTC的具体抑制作用 通过新陈代谢标签。最后，这些抑制剂将在体内使用查加斯病的小鼠模型进行测试。 同时，我们还将在体内测试不同反义寡核苷酸的效果。如果成功，则 拟议的研究将证实我们的假设，即ODTC是设计新药的最佳靶点之一 对抗克氏锥虫。目前，只有一种商业药物可用于治疗恰加斯病。 克氏锥虫耐药株的出现清楚地表明了对新药的迫切需要。 这种影响拉丁美洲数百万人的传染病，是一种紧急公共卫生 在美国引起了担忧。

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.