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Assay Development and Optimization for High Throughput Screen to Detect Compounds Increasing Secretion of C150S Mutant Uromodulin

用于检测增加 C150S 突变体尿调节蛋白分泌的化合物的高通量筛选的测定方法开发和优化

基本信息

批准号：
10534214
负责人：
Matthias Tilmann Florian Wolf
金额：
$ 35.68万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-10 至 2024-12-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT Heterozygous mutations in Uromodulin (UMOD) cause autosomal-dominant tubulo-interstitial kidney disease (ADTKD-UMOD) which results in chronic and end-stage renal disease, and so far no specific treatment is available for these patients. In different murine and cell culture models, it has been found that UMOD mutations impair UMOD protein trafficking. Misfolded UMOD accumulates within the endoplasmic reticulum, resulting in apoptosis of the thick ascending limb (TAL) cells and renal fibrosis. We have established a cell culture-based assay which monitors secretion dynamics of wild-type (WT) and human mutation C150S UMOD in culture medium of stable cell lines expressing luciferase-tagged UMOD plasmids, thereby measuring a disease-relevant endpoint. Our objective for this proposal is to identify and chemically optimize compounds that enhance the secretion of retained mutant UMOD protein as a new therapeutic modality for treating ADTKD-UMOD. Our hypothesis is that increasing secretion of retained, mutant UMOD protein will reduce cellular apoptosis, and will mitigate chronic kidney disease in ADTKD-UMOD. In preliminary data, we screened 8,000 compounds of a subset chemical library for enhancing the secretion of C150S UMOD. We identified five hits that increased C150S UMOD secretion to at least 75% compared to WT UMOD, providing proof-of-concept for our approach. The rationale of this project is to identify potent compounds accelerating secretion of different UMOD mutations using a human kidney cell line to ameliorate ADTKD-UMOD. After screening our established canine C150S UMOD expressing stable cell line against the optimized over 150,000 compounds containing UTSW chemical library, we will counter-screen identified candidates against stably transfected human kidney cell lines expressing the C150S and five other human UMOD mutations. All of these mutations affect one of 24 disulfide bridges, which are involved in up to 60% of UMOD mutations. With this approach, we attempt to treat a maximum number of patients. In aim 1, we will first optimize all plate-based secondary assays including a UMOD ELISA, an assay testing for non-specific upregulation of the secretory pathway, a caspase 3/7 assay, and an assay for testing different UMOD mutations. In aim 2, we will conduct a large phenotypic HTS of the over 150,000 compounds containing UTSW chemical library and will confirm hits in triplicates. A subsequent cell toxicity assay will exclude compounds resulting in false positive hits due to UMOD release caused by significant toxicity. Secondary assays optimized in aim 1 will be performed to decrease the number of promising hits. In aim 3, the best candidates will be tested in a primary cell culture model of the TAL from WT and a mutant Umod mouse model. We will test the stimulatory effect of compounds on mutant UMOD secretion by studying current density of the calcium channel TRPV5. SAR and chemical optimization of the best hits will be studied and tested in the assays outlined in aims 1 and 3. Finally, baseline pharmacology characteristics will be evaluated. Results from these experiments will be significant as they may provide novel and innovative therapeutic options for ADTKD-UMOD patients.

项目摘要/摘要尿调蛋白(UMOD)杂合突变导致常染色体显性遗传性肾小管间质肾病 (ADTKD-UMOD)会导致慢性和终末期肾脏疾病，到目前为止还没有具体的治疗方法适用于这些患者。在不同的小鼠和细胞培养模型中，已经发现UMOD突变阻碍UMOD蛋白的运输。错误折叠的UMOD在内质网内积累，导致粗大升肢(TAL)细胞凋亡与肾脏纤维化我们已经建立了以细胞培养为基础的监测野生型(WT)和人突变C150S UMOD在培养物中分泌动态的方法表达荧光素酶标记的UMOD质粒的稳定细胞系的培养液，从而测量与疾病相关的终结点。我们这项提议的目标是确定并在化学上优化化合物，以增强分泌残存突变的UMOD蛋白作为治疗ADTKD-UMOD的新方法。我们的假说是，增加保留的、突变的UMOD蛋白的分泌将减少细胞凋亡，并将在ADTKD-UMOD中缓解慢性肾脏疾病。在初步数据中，我们筛选了8000种化合物促进C150S UMOD分泌的亚组化学文库。我们确定了五个增加C150S的命中率 UMOD分泌量至少是WT UMOD的75%，为我们的方法提供了概念验证。这个该项目的基本原理是识别有效的化合物，促进不同UMOD突变的分泌改良ADTKD-UMOD的人肾细胞系。在对我们已建立的犬只C150S UMOD进行筛查后对含有UTSW化学文库的150,000多种优化化合物表达稳定的细胞系，我们将从稳定表达该基因的人肾细胞系中反筛选已确定的候选 C150S和其他五个人类UMOD突变。所有这些突变都会影响24个二硫键中的一个，即参与了高达60%的UMOD突变。使用这种方法，我们尝试处理最大数量的病人。在目标1中，我们将首先优化所有基于平板的二次检测，包括UMOD-EL ISA，一种检测检测分泌途径的非特异性上调、caspase3/7检测和用于检测的检测不同的UMOD突变。在目标2中，我们将对超过150,000种化合物进行大型表型HTS 包含UTSW化学库，并将确认命中三份。随后的细胞毒性测试将排除由于UMOD释放引起的显著毒性而导致假阳性的化合物。二次分析将执行目标1中的优化，以减少有希望的命中数量。在目标3中，最好的候选人将在WT的TAL和突变的Umod小鼠模型的原代细胞培养模型中进行测试。我们将测试通过钙通道电流密度研究化合物对突变型UMOD分泌的刺激作用 TRPV5。最佳命中率的合成孔径雷达和化学优化将在AIMS概述的分析中进行研究和测试 1和3。最后，将评估基线药理学特征。这些实验的结果将它们可能为ADTKD-UMOD患者提供新颖和创新的治疗选择，因此具有重要意义。