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: 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
• 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

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.

这项研究的长期目标是调查油酸去饱和酶（或 δ-12去饱和酶）可用作开发新的 化疗药物对抗这种病原体。T. cruzi是南美锥虫病的病原体， ODTc是通过脱氢酶将油酸（C18：1）转化为亚油酸（C18：2）所必需的 反应亚油酸及其下游产物亚麻酸（C18：3）对于保持流动性至关重要 和T.寄生虫一生中温度变化期间的cruzi质膜 周期有趣的是，ODTc在某种意义上是独特的，这种酶或其推定的同源物不存在于 人类因此，它可以作为一个很好的分子靶点，用于治疗目的的T。cruzi 感染为了验证ODTc作为靶标，我们的目标是通过生物化学方法研究这种酶的基本性质。 和分子方法学。在这方面，将采取两项战略。在Aim-1中， 用两种方法沉默ODTc基因的表达。首先，我们将进行淘汰赛 实验第二，使用反义硫代磷酸将下调ODTc的表达 寡核苷酸活力和感染性试验以及生化特性的研究 ODTc无效突变体的研究将为该酶在T.克鲁兹在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δ-6和δ-9抑制剂的结构产生ODTc的选择性抑制剂 去饱和酶将在上鞭毛体和锥鞭毛体中进行药物的高通量筛选 使用改良的存活力（MTT）测定法。下一步将是确认ODTc的特异性抑制 通过代谢标记。最后，将使用恰加斯病的鼠模型在体内测试抑制剂。 并排，我们还将测试不同的反义寡核苷酸在体内的功效。如果成功， 这项研究将验证我们的假设，即ODTc是设计新药的最佳靶点之一 抗犬弓克鲁兹目前，只有一种商业药物可用于治疗南美锥虫病。 T.克氏耐药菌株清楚地指出，迫切需要新的药物， 这种传染病影响了拉丁美洲数百万人，是一种紧急的公共卫生问题， 美国的担忧。