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）：中链酰基辅酶A脱氢酶缺乏症（MCADD）是一种先天性脂肪酸代谢缺陷。 在大多数北方欧洲血统的高加索人中，该疾病的总体频率约为1：12，000。 MCADD患者在出生时是正常的，但存在急性、危及生命的代谢失代偿发作的风险。 这些通常发生在3至24个月的年龄，但可以发生在任何年龄与生理应激，如禁食或感染。 在以前未确诊的患者中，急性危机期间的死亡率可高达20%。 随着通过串联质谱法扩大新生儿筛查的引入，MCADD现在可以在产前进行鉴定，几乎消除了这种疾病的死亡率。 然而，治疗需要终身饮食监测，并且由于在口服摄入量减少的情况下IV葡萄糖治疗的住院治疗仍然会发生显著的发病率。 MCAD基因中的单一突变（G985A点突变）已在缺陷患者中MCAD基因中90%的等位基因中被鉴定。 该突变在成熟酶的位置304（K304E）处用谷氨酸取代赖氨酸，引入四个异常负电荷，使酶的四级结构不稳定。 结果，突变蛋白质被迅速降解。 体外研究表明，突变蛋白质在稳定时具有催化活性。 重要的是，已发表的体内和体外数据表明，仅恢复百分之几的正常MCAD活性将恢复患者接近正常的代谢平衡。 本研究的长期目标是开发治疗MCAD和其他结构相似的酰基辅酶A脱氢酶（ACD）缺陷的分子策略。 本申请的具体目的是通过鉴定可在体外稳定MCAD K304 E突变蛋白的小分子先导化合物，确定MCAD最常见突变体的可药性。 有两个具体目标。 具体目标1的重点是使用两种随机体外化学文库筛选技术，HCS（高内容图像筛选）免疫测定和HTS（高通量筛选）酶法测定，鉴定化学伴侣蛋白，并使用计算机小片段文库筛选鉴定候选化合物。 研究人员最近获得的MCAD K304E突变体的晶体结构将用于设计预测与异常K304E四聚体结合并稳定它的化合物。具体目标2专注于候选化合物的体外测试。 潜在药物的疗效将通过挽救MCAD缺陷型的能力来衡量。 这提供了突变酶的可药用性的强得多的指示，同时为候选化合物的安全性的初始确定提供了更高的标准。 将使用两种体外模型系统检查合成的候选化合物改善K304E突变酶稳定性的能力。 项目叙述：中链酰基辅酶A脱氢酶缺乏症（MCADD）是一种先天性脂肪酸代谢缺陷，患者出生时正常，但有发生急性、危及生命的代谢失代偿的风险。 MCAD基因中的单一突变导致赖氨酸被谷氨酸取代，使酶不稳定并导致其快速降解，已在缺陷患者中90%的MCAD基因中鉴定出。 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，从而降低患者发生危及生命的失代偿和住院的风险，并改善他们的整体生活质量。