Oleate Desaturase: Novel Drug Target for Chagas Disease

油酸去饱和酶：恰加斯病的新药物靶点

基本信息

批准号：
7858090
负责人：
ROSA A MALDONADO
金额：
$ 14.59万
依托单位：
UNIVERSITY OF TEXAS EL PASO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7858090
关键词：
Affect Antisense Oligonucleotides Appearance Architecture Biochemical Biological Assay Cell membrane Chagas Disease Characteristics Communicable Diseases Conversion disorder Data Development Drug Delivery Systems Effectiveness Enzymes Evaluation GPI Membrane Anchors Generations Genes Glycoproteins Glycosylphosphatidylinositols Goals Homologous Gene Human Immune response In Vitro Infection Investigation Knock-out Label Latin America Life Cycle Stages Linoleic Acids Linolenic Acids Lipids Macrophage Activation Membrane Membrane Lipids Metabolic Metabolic Pathway Methodology Modeling Molecular Molecular Target Mucin 1 protein Mucins Mus Nature Oleic Acids Oxidoreductase Parasites Pharmaceutical Preparations Phosphatidylcholine desaturase Phosphorothioate Oligonucleotide Play Polyunsaturated Fatty Acids Property Public Health Reaction Research Role Side Stearoyl-CoA Desaturase Structure Temperature Testing Texas Therapeutic Treatment Efficacy Trypanosoma cruzi United States Universities Validation Variant chemotherapeutic agent desaturase design efficacy testing enzyme activity fluidity high throughput screening in vivo inhibitor/antagonist knockout gene mutant novel pathogen programs research study resistant strain response

项目摘要

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.

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）可作为开发新型锥虫的靶标针对这种病原体的化疗药物。克氏锥虫 (T. cruzi) 是恰加斯病的病原体，表达 ODTc 对于脱氢酶将油酸 (C18:1) 转化为亚油酸 (C18:2) 至关重要反应。亚油酸及其下游产品亚麻酸 (C18:3) 对于保持流动性至关重要以及寄生虫一生中温度变化期间克氏锥虫质膜的结构循环。有趣的是，ODTc 是独特的，因为这种酶或其推定的同系物不存在于人类。因此，它可以作为治疗克氏锥虫的优异分子靶点感染。为了验证 ODTc 作为靶标，我们旨在通过生化研究该酶的本质和分子方法学。在这方面，将采取两种策略。在 Aim-1 中，将尝试使用两种方法来沉默 ODTc 基因的表达。首先，我们将进行淘汰赛实验。其次，使用反义硫代磷酸酯会下调ODTc的表达寡核苷酸。活力和感染性测试以及生化特性的研究 ODTc 无效突变体的分析将提供有关该酶在 T. cruzi 中的作用的重要信息。在目标 2 中， ODTc抑制剂的开发和测试将在体内和体外进行。我们建议通过修改已知的 delta-6 和 delta-9 抑制剂的结构，生成 ODTc 的选择性抑制剂去饱和。将在上鞭毛体和锥鞭毛体中进行药物的高通量筛选使用改良的活力（MTT）测定形成。下一步将确认ODTc的特异性抑制作用通过代谢标记。最后，将使用恰加斯病小鼠模型对抑制剂进行体内测试。同时，我们还将测试不同反义寡核苷酸在体内的功效。如果成功的话，拟议的研究将验证我们的假设，即 ODTc 是设计新药的最佳靶标之一反对 T. cruzi。目前，只有一种商业药物可用于治疗恰加斯病。克氏锥虫耐药菌株的出现清楚地表明迫切需要新的药物来对抗这种传染病影响了拉丁美洲数百万人，是一种突发公共卫生事件美国的担忧。