Podocyte-based HCS assays for discovering therapeutics against kidney diseases

 描述(由申请人提供)：慢性肾脏病(CKD)是美国的一个主要健康问题，在所有成年人中患病率为10%。糖尿病、高血压、肥胖和心脏病是常见的危险因素。CKD目前的治疗选择有限，只能发挥系统作用，目前还没有直接和选择性靶向的药物。 肾脏细胞。足细胞是一种特殊的上皮细胞，对肾脏功能至关重要。足细胞在肾脏中形成滤过屏障，是蛋白尿肾脏疾病的有效细胞靶点。然而，由于缺乏筛查试验，足细胞保护性疗法的合理发展受到了极大的阻碍。我们最近开发了一种基于足细胞的新的筛选方法，并对其进行了优化，用于96孔板。我们的基于图像的分析显示，在两个独立的初级分析读数中，Z‘值分别为0.46和0.44，对于多参数分类器分析读数，Z’&gt；值为0.65。在筛选了&gt；2100种不同化学物质的生物活性文库后，我们确定了24种Hit，在二次和功能体外测试中验证了它们，并在已建立的蛋白尿肾病体内模型中验证了一种选择Hit。对损伤的特定分子标记的分析也为我们提供了一种新的HIT化合物的潜在作用机制。这些数据表明，我们的新方法可能是对足细胞进行合理的化学文库分析的基础。因此，在这里，我们建议我们的基于细胞的表型分析可以进一步微型化，并应用于高温超导环境中，以识别保护足细胞免受损伤的新的小分子。此外，已鉴定的HIT可以在现有的体外和体内下游检测中得到验证。我们的长期目标是开发新的足细胞保护化合物，用于治疗人类各种蛋白尿性肾脏疾病。我们提出的三个目标包括：1)用于HTS环境的新开发的基于足细胞的检测方法的实施和微型化，2)使用新的检测方法执行HTS活动，然后确认和优先处理HIT化合物，以及3)使用体外和体内测试验证识别的HITS。我们已经有了所有的体外和体内分析方法，并在实验室进行了验证。我们还在拉什和桑福德-伯纳姆组建了一个专家团队，成功地实现了我们提出的目标。我们希望我们提出的研究，如果成功，将导致识别新的化学探针和化合物，直接针对足细胞并保护它们免受伤害。我们还期待所鉴定的化合物在未来可以进一步发展成为治疗人类蛋白尿肾病的药物，从而改善CKD患者的生活。

 DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is a major health problem in the US, with prevalence of >10% among all adults. Diabetes, hypertension, obesity and heart disease are common risk factors. CKD is currently managed with limited therapeutic options that act systemically and there are currently no drugs that directly and selectively target the kidney cells. Podocytes are specialized epithelial cells that are central for kidney function. Podocytes form the filtration barrier in the kidney and are a validated cellular target for proteinuric kidney diseases. Yet, rational development of podocyte protective therapeutics has been greatly hampered by lack of screening assays. We recently developed a novel podocyte cell-based screening assay and optimized it for use in 96-well plates. Our image-based assay showed Z' values of 0.46 and 0.44 in two independent primary assay readouts and a Z' > 0.65 for a multi-parametric classifier assay readout. Upon screening of a bioactive library of >2100 diverse chemicals, we identified 24 hits, validated them in secondary and functional in vitro assays and validated one select hit in an established in vivo model of proteinuric kidney disease. Analysis of specific molecular markers of injury also provided us with a potential mechanism of action of the novel hit compound. These data suggest that our novel assay could be a basis for rational chemical library profiling on podocytes. Thus, here, we propose that our cell based phenotypic assay could be further miniaturized and applied in an HTS environment to identify novel small molecules that protect podocytes from injury. Additionally, the identified hit could be validated in available downstream in vitro and in vivo assays. Our long-term goal is to develop novel podocyte- protective compounds for the treatment of a variety of proteinuric kidney diseases in humans. Our three proposed aims include: 1) Implementation and miniaturization of a newly developed podocyte cell-based assay for use in an HTS environment, 2) Execution of HTS campaign using the novel assay followed by confirmation and prioritization of the hit compounds, and 3) Validation of identified hits using in vitro and in vivo assays. We have all the in vitro and in vivo assays in place and validated in the laboratory. We have also assembled a team of experts at Rush and at Sanford-Burnham to successfully implement our proposed aims. We expect that our proposed studies, if successful, will lead to identification of novel chemical probes and compounds that directly target podocytes and protect them from injury. We also expect that the identified compounds could be further developed into therapeutics for treating proteinuric kidney disease in humans in the future, thus improving lives of patients with CKD.