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，在所有成年人中的患病率>10%。糖尿病、高血压、肥胖和心脏病是常见的风险因素。CKD目前的治疗选择有限，系统性作用，目前没有直接和选择性靶向药物 肾脏细胞足细胞是特化的上皮细胞，其对于肾功能至关重要。足细胞形成肾脏中的过滤屏障，并且是蛋白尿性肾病的经验证的细胞靶标。然而，由于缺乏筛选试验，足细胞保护疗法的合理开发受到极大阻碍。我们最近开发了一种新的足细胞为基础的筛选试验，并优化其用于96孔板。我们的基于图像的测定在两个独立的初级测定读出中显示出0.46和0.44的Z'值，并且对于多参数分类器测定读出，Z' > 0.65。在筛选了>2100种不同化学物质的生物活性文库后，我们鉴定了24个命中，在二级和功能性体外测定中验证了它们，并在建立的蛋白尿肾病体内模型中验证了一个选择的命中。对损伤的特异性分子标记物的分析也为我们提供了新命中化合物的潜在作用机制。这些数据表明，我们的新的测定法可能是合理的足细胞化学库分析的基础。因此，在这里，我们提出，我们的细胞为基础的表型测定可以进一步小型化，并在HTS环境中应用，以确定新的小分子，保护足细胞免受损伤。此外，可以在可用的下游体外和体内测定中验证鉴定的命中。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病。我们提出的三个目标包括：1）在HTS环境中使用的新开发的基于足细胞的测定法的实施和小型化，2）使用新测定法执行HTS活动，然后确认和优先化命中化合物，和3）使用体外和体内测定法验证鉴定的命中。我们拥有所有的体外和体内试验，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小组，以成功实现我们提出的目标。我们希望我们提出的研究，如果成功的话，将导致新的化学探针和化合物的鉴定，直接靶向足细胞，并保护他们免受伤害。我们还期望，所鉴定的化合物可以在未来进一步开发成治疗人类蛋白尿性肾病的疗法，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.