Development of Chemical Chaperonin for Medium Chain Acyl-CoA Dehydrogenase Defici

针对中链酰基辅酶A脱氢酶缺乏症的化学伴侣蛋白的开发

• 批准号: 7738575
• 负责人: Al-Walid Abdel Mohsen
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-05 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7738575
• 关键词: Acute; Acyl CoA Dehydrogenases; Affinity; Age; Age-Months; Alleles; Amides; Antibodies; Antigens; Binding; Biochemical Genetics; Biological Assay; Biological Models; Birth; Caliber; Caucasians; Caucasoid Race; Cells; Charge; Chemicals; Cleaved cell; Complex; Computer Simulation; Data; Descriptor; Development; Diet Monitoring; Disease; Electron transfer flavoprotein; Enzyme Stability; Enzymes; Equilibrium; Esters; European; Face; Family suidae; Fasting; Fever; Fibroblasts; Frequencies; Genes; Glucose; Glutamates; Hereditary Disease; Hospitalization; Human; Human Activities; Hydrogen Bonding; Image; Immunoassay; In Vitro; Inborn Genetic Diseases; Infection; Intake; Ions; Lead; Libraries; Life; Lymphocyte; Lysine; Measures; Metabolic; Molecular; Molecular Models; Morbidity - disease rate; Mutation; Neonatal Screening; Oral; Oxidants; Oxygen; Patients; Pharmaceutical Preparations; Phase; Phenotype; Phenylalanine Hydroxylase; Physiological; Point Mutation; Positioning Attribute; Process; Proteins; Publishing; Quality of life; Research Personnel; Risk; Safety; Screening procedure; Side; Signal Transduction; Site; Stress; Structure; Study models; System; Techniques; Testing; Toxic effect; United States; Western Blotting; acyl-CoA dehydrogenase; acylcarnitine; amino group; base; candidate identification; chaperonin; design; dimer; drug candidate; enzyme structure; fatty acid metabolism; high throughput screening; improved; in vitro Model; in vitro testing; in vivo; molecular modeling; monomer; mortality; mutant; octanoyl-coenzyme A; orphan disease/drug; protein folding; restoration; small molecule; small molecule libraries; tandem mass spectrometry; time use; treatment strategy

DESCRIPTION (provided by applicant): Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inborn error of fatty acid metabolism. The overall frequency of the disease is ~1:12,000 in Caucasians of mostly Northern European ancestry. MCADD patients are normal at birth but are at risk for episodes of acute, life threatening metabolic decompensation. These usually occur between three and twenty four months of age but can occur at any age in association with physiologic stress such as fasting or infection. The mortality rate during an acute crisis in previously undiagnosed patients can be as high as 20%. With the introduction of expanded newborn screening via tandem mass spectrometry, MCADD can now be identified pre-symptomatically, nearly eliminating mortality due to this disease. However, treatment requires lifelong dietary monitoring, and significant morbidity still occurs due to hospitalizations for IV glucose therapy in the face of reduced oral intake. A single mutation in the MCAD gene (a G985A point mutation) has been identified in 90% of the alleles in the MCAD gene in deficient patients. This mutation substitutes a glutamate for a lysine at position 304 of the mature enzyme (K304E), introducing four abnormal negative charges, destabilizing the quaternary structure of the enzyme. As a result, the mutant protein is rapidly degraded. In vitro studies have shown that the mutant protein is catalytically active when it can be stabilized. Importantly, published in vivo and in vitro data suggest that restoration of only a few percent of normal MCAD activity will restore near normal metabolic balance in patients. The long range objective of this study is to develop molecular strategies for treatment of deficiencies of MCAD and other structurally similar acyl-CoA dehydrogenases (ACDs). The specific objective of this application is to establish the drugability of the MCAD most common mutant by identifying small molecule lead compounds that can stabilize the MCAD K304E mutant protein in vitro. There are two specific aims. Specific Aim 1 is focused on the identification of chemical chaperonins using two random in vitro chemical library screening techniques, an HCS (high content-image based screening) immunoassay and HTS (high throughput screening) enzymatic assay, and to identify candidate compounds using in silico small fragments library screening. The crystal structure of MCAD K304E mutant the investigators obtained recently will be used to design compounds that are predicted to bind to the abnormal K304E tetramer and stabilize it. Specific Aim 2 is focused on the in vitro testing of candidate compounds. Efficacy of potential drugs will be measured by the ability to rescue the MCAD deficient phenotype. This provides a much stronger indication of the drugability of the mutant enzyme while providing a higher bar for initial determination of safety of candidate compounds. Synthesized candidate compounds will be examined for their ability to improve stability of the K304E mutant enzyme using two in vitro model systems. PROJECT NARRATIVE: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inborn error of fatty acid metabolism with patients found to be normal at birth but are at risk for episodes of acute, life threatening metabolic decompensation. A single mutation in the MCAD gene, which causes the replacement of a lysine with a glutamate destabilizing the enzyme and resulting in its rapid degradation, has been identified in 90% of the MCAD gene in deficient patients. The long term objective of this project is to develop a chemical chaperonin for stabilizing the MCAD enzyme, using in vitro and in silico approaches, and hence reduce patients' risk for life threatening episodes of decompensation and hospitalization, and improve their overall quality of life.

描述（由申请人提供）：中链酰基-COA脱氢酶缺乏症（MCADD）是脂肪酸代谢的天生错误。 该疾病的总频率为〜1：12,000，主要是北欧血统的高加索人。 MCADD患者出生时正常，但有急性，威胁生命的代谢代谢的风险。 这些通常发生在三到二十四个月之间，但可以在任何年龄段与生理压力（例如禁食或感染）相关。 先前未诊断的患者急性危机期间的死亡率高达20％。 随着通过串联质谱进行扩展的新生儿筛查的引入，现在可以在遗传上识别McAdd，几乎消除了由于这种疾病而导致的死亡率。 但是，治疗需要终生饮食监测，并且由于口服摄入量减少而导致静脉葡萄糖疗法的住院治疗导致明显的发病率。 在不足患者中，MCAD基因的90％等位基因中已经发现了MCAD基因中的单个突变（G985a点突变）。 该突变将谷氨酸取代成熟酶（K304E）位置304的赖氨酸，引入了四个异常的负电荷，破坏了酶的季季结构。 结果，突变蛋白迅速降解。 体外研究表明，突变蛋白可以稳定时具有催化活性。 重要的是，在体内和体外数据中发表的数据表明，仅恢复正常的MCAD活性的几％将恢复患者的代谢平衡接近正常的代谢平衡。 这项研究的远程目标是制定分子策略，用于治疗MCAD和其他结构相似的酰基-COA脱氢酶（ACD）的缺陷。 该应用的具体目标是通过鉴定可以在体外稳定MCAD K304E突变蛋白稳定的小分子铅化合物来确定MCAD最常见突变体的可药用性。 有两个具体的目标。 特定的目标1专注于使用两种随机体外化学库筛选技术鉴定化学伴侣蛋白，HC（基于高含量图像图像的筛选）免疫测定法和HTS（高吞吐量筛选）酶法测定法，以及使用硅胶小片段库筛选中的候选化合物鉴定候选化合物。 MCAD K304E突变体的晶体结构最近获得的研究者将用于设计与异常K304E四聚体结合并稳定它的化合物。 特定的目标2集中于候选化合物的体外测试。 潜在药物的功效将通过挽救MCAD缺乏表型的能力来衡量。 这为突变酶的可药用性提供了更大的迹象，同时为候选化合物的安全性提供了更高的标准。 将检查合成的候选化合物，以使用两个体外模型系统来提高K304E突变酶的稳定性。 项目叙述：中链酰基-COA脱氢酶缺乏症（MCADD）是脂肪酸代谢的天生误差，患者发现出生时正常，但有急性发作，威胁生命的新陈代谢失代偿性的风险。 MCAD基因中的单个突变导致赖氨酸替代了谷氨酸，使酶不稳定并导致其快速降解，在缺乏患者的90％的MCAD基因中已鉴定出其快速降解。 该项目的长期目标是开发一种化学伴侣蛋白，用于使用体外和计算机方法稳定MCAD酶，从而降低患者发生威胁生命的代偿性和住院发作的风险，并改善其整体生活质量。