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

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错误。在大多数北欧血统的高加索人中，该病的总体发病率约为1:12 000。MCADD患者在出生时是正常的，但有急性、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个月大之间，但也可能发生在任何年龄，与禁食或感染等生理压力有关。以前未确诊的患者在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现在可以在症状前被识别出来，几乎消除了这种疾病造成的死亡率。然而，治疗需要终生的饮食监测，在口服葡萄糖摄入量减少的情况下，仍然会因静脉葡萄糖治疗住院而发生显著的发病率。在缺乏MCAD的患者中，在90%的MCAD基因等位基因中发现了单个突变（G985A点突变）。该突变将成熟酶（K304E） 304位的赖氨酸替换为谷氨酸，引入了四个异常负电荷，破坏了酶的四级结构。结果，突变蛋白被迅速降解。体外研究表明，突变蛋白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，仅恢复正常MCAD活性的百分之几就可以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和其他结构类似的酰基辅酶a脱氢酶（ACDs）缺陷的分子策略。本申请的具体目的是通过鉴定可以在体外稳定MCAD K304E突变蛋白的小分子先导化合物来确定MCAD最常见突变体的可药物性。有两个具体目标。Specific Aim 1的重点是使用两种随机的体外化学文库筛选技术，HCS（高含量图像筛选）免疫测定和HTS（高通量筛选）酶促测定来鉴定化学伴侣蛋白，并使用硅小片段文库筛选来鉴定候选化合物。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四聚体结合并稳定其的化合物。特异性目标2侧重于候选化合物的体外测试。潜在药物的疗效将通过挽救MCAD缺陷表型的能力来衡量。这为突变酶的可药物性提供了更强的指示，同时为初步确定候选化合物的安全性提供了更高的标准。合成的候选化合物将使用两种体外模型系统检查其提高K304E突变酶稳定性的能力。

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.