Oleate Desaturase: Novel Drug Target for Chagas Disease

油酸去饱和酶：恰加斯病的新药物靶点

• 批准号: 8080330
• 负责人: ROSA A MALDONADO
• 金额: $ 12.57万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8080330
• 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; Therapeutic; Treatment Efficacy; Trypanosoma cruzi; United States; Validation; Variant; chemotherapeutic agent; desaturase; design; efficacy testing; enzyme activity; fluidity; high throughput screening; in vivo; inhibitor/antagonist; knockout gene; mutant; novel; pathogen; research study; resistant strain; response

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研究酶Oleate去饱和酶是否（或 Cruzi锥虫（ODTC）的Delta-12去饱和酶）可以用作发展新颖的靶标 反对这种病原体的化学治疗剂。 Chagas疾病的病因学家T. Cruzi表示 ODTC对于通过脱氢酶将油酸（C18：1）转化为亚油酸（C18：2）的ODTC是必不可少的 反应。亚油酸及其下游产物亚麻酸（C18：3）对于维持流动性至关重要 以及在整个寄生虫寿命中温度变化期间T. cruzi质膜的结构 循环。有趣的是，ODTC是独一无二的，从某种意义上说，这种酶或其假定的同源物不存在 人类。因此，它可以作为针对克鲁兹的治疗目的的极好的分子靶标 感染。为了验证ODTC为目标，我们旨在通过生化研究该酶的基本性质 和分子方法。在这方面，将采取两种策略。在AIM-1中，将尝试 使用两种方法使ODTC基因的表达保持沉默。首先，我们将执行淘汰赛 实验。其次，使用反义磷脂酸盐将ODTC的表达下调 寡核苷酸。生存力和感染性的测试以及生化特性的研究 ODTC无效突变体将提供有关该酶在T. Cruzi中作用的重要信息。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 通过修改Delta-6和Delta-9的已知抑制剂的结构来生成ODTC的选择性抑制剂 去饱和酶。将在Epimastigote和Trypomastigote中对药物进行高通量筛查 使用修改的生存力（MTT）测定法。下一步将是确认ODTC的特定抑制 通过代谢标签。最后，抑制剂将在体内使用Chagas疾病的鼠模型在体内进行测试。 同时，我们还将测试体内不同反义寡核苷酸的功效。如果成功， 拟议的研究将验证我们的假设，即ODTC是设计新药的最佳目标之一 对阵T. Cruzi。目前，只有一种商业药物可用于治疗木马疾病。 抗克氏菌抗菌株的外观清楚地指出了迫切需要对 这种传染病影响了拉丁美洲数百万的人，这是一种新兴的公共卫生 在美国的关注。