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

• 批准号: 10311119
• 负责人: Matthias Tilmann Florian Wolf
• 金额: $ 36.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10311119
• 关键词: Affect; Apoptosis; Biochemistry; Biological Assay; CASP3 gene; Calcium Channel; Canis familiaris; Cell Culture Techniques; Cell Line; Cell secretion; Cells; Characteristics; Chemicals; Chemistry; Chronic; Chronic Kidney Failure; Clinical Pharmacology; Complement; Core Facility; Data; Defect; Dialysis procedure; Disease; Disulfides; Embryo; End stage renal failure; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Fibrosis; Functional disorder; Future; Genetic; Goals; Human; Human Cell Line; Impairment; Inflammation; Kidney; Kidney Diseases; Kidney Failure; Kidney Transplantation; Lead; Libraries; Limb structure; Luciferases; MDCK cell; Measures; Metabolism; Mission; Modality; Modeling; Molecular; Molecular Target; Monitor; Mus; Mutation; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Pharmacology; Phenotype; Plasmids; Polycystic Kidney Diseases; Primary Cell Cultures; Protein Secretion; Proteins; Reporter; Research; Solubility; Structure-Activity Relationship; Supportive care; Testing; Therapeutic; Thick; Toxic effect; Tubular formation; UMOD gene; United States National Institutes of Health; Up-Regulation; Wolves; analog; assay development; base; counterscreen; density; disulfide bond; experimental study; high throughput screening; improved; improved outcome; in vivo; innovation; interstitial; kidney cell; kidney fibrosis; misfolded protein; molecular pathology; mouse model; mutant; novel; novel therapeutics; patch clamp; pre-clinical; prevent; protein transport; scaffold; screening; small molecule libraries; stable cell line; tool; trafficking; translational impact

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.

项目总结/摘要 尿调蛋白（UMOD）杂合突变导致常染色体显性肾小管间质肾病 ADTKD-UMOD），其导致慢性和终末期肾病，并且迄今为止没有特异性治疗。 为这些患者提供。在不同的鼠和细胞培养模型中，已经发现UMOD突变 削弱UMOD蛋白运输。错误折叠的UMOD在内质网内积累，导致 粗升支（TAL）细胞凋亡和肾纤维化。我们建立了一个基于细胞培养的 监测培养物中野生型（WT）和人突变C150 S UMOD分泌动力学的测定 在表达加有谷胱甘肽转移酶标记的UMOD质粒的稳定细胞系的培养基中， 终点。我们的目标是确定和化学优化化合物，提高 作为治疗ADTKD-UMOD的新治疗方式的保留突变UMOD蛋白的分泌。我们 假设是增加保留的突变UMOD蛋白的分泌将减少细胞凋亡， 缓解ADTKD-UMOD中的慢性肾病。在初步数据中，我们筛选了8,000种化合物， 用于增强C150 S UMOD分泌的亚组化学文库。我们发现了五个能增加C150 与WT UMOD相比，UMOD分泌至少为75%，为我们的方法提供了概念验证。的 该项目的基本原理是使用以下方法鉴定加速不同UMOD突变分泌的有效化合物： 改善ADTKD-UMOD的人肾细胞系。在筛选我们建立的犬C150 S UMOD后， 针对优化的超过150，000种包含UTSW化学文库的化合物表达稳定的细胞系， 我们将针对稳定转染的人肾细胞系反筛选鉴定的候选物， C150 S和其他五种人类UMOD突变。所有这些突变都会影响24个二硫键中的一个， 与高达60%的UMOD突变有关。通过这种方法，我们试图治疗最大数量的 患者在目标1中，我们将首先优化所有基于平板的二级检测，包括UMOD ELISA， 测试分泌途径的非特异性上调、半胱天冬酶3/7测定和测试测定 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化学文库，并将一式三份确认命中。随后的细胞毒性试验将排除 由于显著毒性引起的UMOD释放，化合物导致假阳性命中。二次检测 将执行目标1中的优化以减少有希望的命中的数量。在目标3中，最佳候选人将 在来自WT的TAL的原代细胞培养模型和突变体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来研究化合物对突变体UMOD分泌的刺激作用 TRPV 5。最佳命中的SAR和化学优化将在目标中概述的测定中进行研究和测试。 1和3.最后，将评价基线药理学特征。这些实验的结果将 因为它们可能为ADTKD-UMOD患者提供新的和创新的治疗选择。