Small Molecule KLF15 Agonists for Kidney Disease

疾病控制和预防中心估计，美国有超过15%的成年人 在美国，超过3000万美国人患有慢性肾脏病（CKD）。足细胞是足中的上皮细胞， 肾小球，其主要功能是维持肾脏过滤屏障以预防CKD。 此外，退伍军人人群中CKD的患病率比普通人群高三分之一。 足细胞损伤与诸如局灶节段性肾小球硬化（FSGS）的疾病有关。在这些 当疾病发生时，足细胞失去特征性的形态特征和维持足细胞的功能能力。 肾小球滤过屏障在最近的几项研究中，我们报道了Krüppel样因子15的重要作用， （KLF 15），一种肾富集的转录因子，在细胞应激下维持足细胞肌动蛋白细胞骨架。 例如，在临床前蛋白尿小鼠模型中进行的功能丧失研究表明，KLF 15是 需要防止足细胞损伤和糖皮质激素（GC）的有益作用， 治疗原发性肾小球病的药物，是由KLF 15介导的。同样，在人类中对GC的反应性 原发性肾小球病与足细胞特异性表达KLF 15有关。此外，诱导 足细胞中的人KLF 15减轻了蛋白尿小鼠模型的肾损伤，而没有不利的 GC的后遗症总之，这些关于KLF 15肾保护作用的临床前和临床研究 诱导促使我们鉴定用于肾脏疾病的新型小分子KLF 15激动剂。 我们最初产生并进行了基于细胞的高通量筛选（HTS），以筛选小的 诱导KLF 15活性的分子。随后的剂量递增研究确定了新的先导化合物 在诱导KLF 15所需的最佳治疗窗内，半数最大有效浓度（EC 50） 活动以EC 50和药敏性为基础，对先导化合物进行构效关系（SAR 化合物K-7，并产生了16个先导类似物，其中BT501，BT502，BT503，BT514和BT412诱导 KLF 15启动子活性与或不与细胞应激。我们还进行了K-7的初步药代动力学研究， 还表明用K-7和铅类似物处理的人足细胞减轻了足细胞损伤 in the setting设置of cell细胞stress应力.此外，K-7处理的人足细胞的RNA-seq显示抑制途径 IL-17 RA介导的肌动蛋白细胞骨架不稳定，从而提供了利用一种机制的基本原理。 优化KLF 15激动剂选择性的方法。最后，我们观察到K-7减少了蛋白尿， 在临床前蛋白尿小鼠模型中恢复足细胞标志物。基于这些令人信服的初步证据 数据和先前研究的强大科学严谨性，我们假设， 临床前研究将作为蛋白尿肾病的关键治疗方法。我们建议测试我们的 通过（1）改善先导KLF 15激动剂的药效学和药代动力学特性，（2） 利用机制方法优化KLF 15激动剂的选择性，和（3）测试治疗作用 在临床前蛋白尿模型中，KLF 15激动剂在减轻和/或逆转肾损伤中的作用。 该提案将通过制定一个综合框架， 并在临床前蛋白尿模型中测试其治疗作用。远景目标 我们的项目的目的是确定最佳的KLF 15激动剂，可以推进IND研究，用于治疗 原发性肾小球病识别治疗蛋白尿疾病的新靶点是主要的 考虑到美国退伍军人中CKD的高负担，退伍军人事务部对此很感兴趣。此外，治疗策略 减少GC的长期使用将对并发症产生巨大影响， 美国退伍军人中的GC。最后，KLF 15诱导的治疗作用可能超越肾脏疾病， 其他实验室已经证明了KLF 15在心脏肥大中的有益作用， 神经变性疾病和脂肪生成。

The Centers for Disease Control and Prevention estimates more than 15% of adults in the United States, over 30 million Americans have chronic kidney disease (CKD). Podocytes are epithelial cells in the glomerulus whose major function is the maintenance of the kidney filtration barrier to prevent CKD. Furthermore, the prevalence of CKD in the Veteran population is a third higher than in the general population. Podocyte injury is implicated in diseases such as Focal Segmental Glomerular Sclerosis (FSGS). In these diseases, the podocyte loses characteristic morphologic features and the functional capacity to maintain the glomerular filtration barrier. In several recent studies, we reported the essential role of Krüppel-Like Factor 15 (KLF15), a kidney-enriched transcription factor, in maintaining podocyte actin cytoskeleton under cell stress. For instance, loss of function studies in preclinical proteinuric murine models demonstrated that KLF15 is required to prevent podocyte injury and the salutary benefits of glucocorticoids (GCs), the most common treatment for primary glomerulopathies, are mediated by KLF15. As well, the responsiveness to GCs in human primary glomerulopathies is associated with podocyte-specific expression of KLF15. Furthermore, induction of human KLF15 in podocytes attenuated kidney injury in proteinuric murine models, without the adverse sequelae of GCs. Collectively, these preclinical and clinical studies on the renoprotective effects of KLF15 induction motivated us to identify novel small molecule KLF15 agonists for kidney disease. We initially generated and conducted a cell-based high-throughput screening (HTS) to screen small molecules that induce KLF15 activity. Subsequent dose-escalating studies identified novel lead compounds with a half maximal effective concentration (EC50), in the optimal therapeutic window, required to induce KLF15 activity. Based on EC50 and druggability, we conducted Structure-Activity Relationship (SAR) on the lead compound K-7 and generated 16 lead analogues, of which BT501, BT502, BT503, BT514, and BT412 induced KLF15 promoter activity with or without cell stress. We also performed intial pharmacokinetic studies for K-7 in mice and also showed that human podocytes treated with K-7 and lead analogues attenuated podocyte injury in the setting of cell stress. Furthemore, RNA-seq of K-7 treated human podocytes shows inhibition of pathway IL-17RA-mediated actin cytoskeleton destabilization, thereby providing the rationale to utilize a mechanistic approach to optimize selectivity of KLF15 agonists. Finally, we observed that K-7 attenuated albuminuria and restored podocyte markers in a preclinical proteinuric murine model. Based on these compelling preliminary data and strong scientific rigor of prior research, we hypothesize that optimization of lead KLF15 agonists in preclinical studies will serve as a key therapeutic in proteinuric kidney diseases. We propose to test our hypothesis by (1) improving the pharmacodynamic and pharmacokinetic properties of lead KLF15 agonists, (2) utilizing a mechanistic approach to optimize selectivity of KLF15 agonists, and (3) testing the therapeutic role of lead KLF15 agonists in mitigating and/or reversing kidney injury in preclinical proteinuric models. This proposal will address a current gap in the field by developing an integrated framework to optimize lead novel KLF15 agonists and test their therapeutic role in preclinical proteinuric models. The long-term goal of our project is to identify the optimal KLF15 agonist that can be advanced for IND studies for the treatment of primary glomerulopathies. Identification of novel targets for the treatment of proteinuric diseases is of major interest to the VA, given the high burden of CKD among U.S. Veterans. Furthermore, therapeutic strategies that mitigate the long-term use of GCs will have a tremendous impact on the complications associated with GCs in U.S. Veterans. Finally, the therapeutic role of KLF15 induction might extend beyond kidney disease, as other laboratories have demonstrated the beneficial effects of KLF15 in cardiac hypertrophy, neurodegenerative disease, and adipogenesis.