Using HTS to Identify Inhibitors of R155H-p97/VCP Mutant to treat IBMPFD/ALS

项目摘要 P97 AAA ATPase(也称为VCP、CDC48、TER ATPase)的单一氨基酸突变导致 常染色体显性遗传性人类疾病包括包涵体肌病，Paget病， 额颞叶痴呆(IBMPFD)和肌萎缩侧索硬化症(ALS)等。最常见的 P97突变(R155H)目前占这种罕见的渐进性衰弱的发病率的一半 无法治愈的致命疾病。来自IBMPFD/ALS患者和小鼠模型的强有力的数据表明 针对疾病-p97的突变将导致治疗。由于野生型(WT)p97及其众多 泛素结合辅因子调节蛋白质底物功能，WT p97‘S必备功能 为了人类健康，保持完好无损。以往的研究表明，R155H-p97对 抑制剂，更高的基础ATPase活性，并由相互作用的蛋白质进行差异调节。这表明 这种突变体选择性的小分子可以被鉴定出来，它将抑制R155H-p97，而不影响野生型 打字。这个项目的目标是确定那些具有突变选择性的药物候选小分子。 治疗或预防IBMPFD/ALS病理的发展。为了实现我们的目标，我们提出了三个具体目标。 目的1：鉴定抑制R155H的化合物，R155H是引起IBMPFD/ALS的最常见的p97突变。 在R155H-P97中试筛选期间，开发并验证了高通量筛选的条件 国家高级翻译科学中心(NCATS)2400种药物收藏 批准的药物。该项目将进行500,000种不同的定量高通量筛选(QHTS) R155H-p97抑制剂在p47(p97的主要辅因子之一)存在下的化合物 与NCATS的合作。这些化合物将根据选择性和效力进行排序。目标 2：在一系列生化和基于细胞的分析中测试目标1中确定的化合物，以确保 这些化合物在细胞内和细胞外都具有相同的效果。这些化合物将被描述为 在建立和验证的基于细胞的分析中，监测七个潜在的IBMPFD/ALS的表达 疾病生物标志物。最后，目标3：使用目标2中出现的先导化合物来评估它们是否 疾病小鼠和健康小鼠成纤维细胞中正确的疾病 IBMPFD/ALS和健康人。这些数据以及对血液、脑屏障通透性的评估 应该为进一步的治疗开发确定最好的候选者。

Project Summary Single amino acid mutations in p97 AAA ATPase (also known as VCP, CDC48, TER ATPase) cause autosomal dominant human disorders embracing inclusion body myopathy, Paget's disease of bone, frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS) among others. The most common p97 mutation (R155H) accounts for half the incidence of this rare, progressively debilitating, currently untreatable, fatal disorder. Strong data from IBMPFD/ALS human patients and mouse models suggests that targeting the disease-mutations of p97 will lead to a treatment. Since wild type (WT) p97 and its many ubiquitin-binding cofactors regulate the functions of protein substrates, WT p97’s essential functions must remain intact for human health. Previous studies demonstrated that R155H-p97 has differential sensitivity to inhibitors, higher basal ATPase activity, and is differentially regulated by interacting proteins. This suggests that mutant-selective small molecules can be identified which will inhibit R155H-p97, without effects on wild type. The goal of this project is to identify those mutant-selective small molecule candidates for drug development to treat or prevent IBMPFD/ALS pathology. Three specific aims are proposed to achieve our goal. AIM 1: Identify compounds that inhibit R155H, the most common p97 mutant causing IBMPFD/ALS. Conditions for high-throughput screening were developed and validated during a R155H-p97 pilot screen with the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection of 2,400 approved drugs. This project will carry out a quantitative high-throughput screening (qHTS) of 500,000 diverse compounds for inhibitors of R155H-p97 in the presence of p47 (one of the major cofactors of p97) in collaboration with the NCATS. The compounds will be ranked according to selectivity followed by potency. AIM 2: Test the compounds identified in AIM 1 in a cascade of biochemical and cell-based assays to ensure that the compounds will have the same effect inside as well as outside cells. The compounds will be characterized in established and validated cell-based assays to monitor the expression of seven potential IBMPFD/ALS disease biomarkers. Finally AIM 3: Use the lead compounds that emerge from Aim 2 to evaluate whether they correct disease in fibroblasts obtained from diseased and healthy mice and both human subjects with IBMPFD/ALS and healthy individuals. This data along with evaluation of Blood, Brain Barrier permeability should identify best candidates for further therapy development.