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/摘要尿素结合蛋白（UMOD）的杂合突变引起常染色体显性tubulo Interstitial肾脏疾病（ADTKD-UMOD）导致慢性和终末期肾脏疾病，到目前为止，还没有具体治疗可用于这些患者。在不同的鼠和细胞培养模型中，已经发现UMOD突变损害UMOD蛋白运输。错误折叠的UMOD积聚在内质网中，导致较厚的升肢（TAL）细胞和肾纤维化的凋亡。我们已经建立了基于细胞培养的在培养中监视野生型（WT）和人类突变C150的分泌动力学的测定表达荧光素酶标记的UMOD质粒的稳定细胞系的培养基，从而测量与疾病相关的端点。我们对该建议的目标是确定并化学优化化合物，以增强该化合物保留突变蛋白的分泌是一种用于治疗ADTKD-UMOD的新治疗方式。我们的假设是，保留的，突变的UMOD蛋白的分泌增加会减少细胞凋亡，并将减轻ADTKD-UMOD的慢性肾脏疾病。在初步数据中，我们筛选了8,000种化合物子集化学库，用于增强C150S UMOD的分泌。我们确定了五个命中，增加了C150S 与WT UMOD相比，UMOD分泌至少75％，为我们的方法提供了概念验证。这该项目的基本原理是鉴定有效的化合物使用不同的UMOD突变加速分泌人类肾细胞系可改善ADTKD-UMOD。筛选我们已建立的犬C150S UMOD之后表达稳定的细胞系，以优化超过150,000种包含UTSW化学库的化合物，我们将在屏幕上识别候选者，反对稳定转染的人类肾细胞系，表达 C150和其他五个人类UMOD突变。所有这些突变都会影响24个二硫键之一，这是参与多达60％的UMOD突变。通过这种方法，我们试图治疗最大数量患者。在AIM 1中，我们将首先优化所有基于板的次级测定法，包括UMOD ELISA，一个测定测试分泌途径的非特异性上调，caspase 3/7分析和测试测定法不同的UMOD突变。在AIM 2中，我们将进行超过150,000种化合物的大型表型HTS 包含UTSW化学库，并将确认一式三份的打击。随后的细胞毒性测定将排除由于严重毒性引起的UMOD释放而导致假阳性的化合物。次要测定在AIM 1中进行了优化，以减少有希望的命中次数。在AIM 3中，最好的候选人将在WT的TAL的原代细胞培养模型和突变的UMOD小鼠模型中进行测试。我们将测试通过研究钙通道的电流密度，化合物对突变体UMOD分泌的刺激作用 TRPV5。 SAR和最佳命中的化学优化将在AIMS中概述的测定中进行研究和测试 1和3。最后，将评估基线药理学特征。这些实验的结果将重要，因为它们可能为ADTKD-UMOD患者提供新颖而创新的治疗选择。