Small Molecule KLF15 Agonists for Kidney Disease

疾病控制和预防中心估计，超过15%的美国成年人 在美国，超过3000万美国人患有慢性肾脏疾病(CKD)。足细胞是足细胞中的上皮细胞 肾小球，其主要功能是维持肾滤过屏障，预防CKD。 此外，退伍军人中慢性肾脏病的患病率比普通人群高出三分之一。 足细胞损伤与局灶性节段性肾小球硬化(FSGS)等疾病有关。在这些 疾病，足细胞失去了特有的形态特征和维持 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的基本作用 (KLF15)，一个肾脏丰富的转录因子，在细胞应激下维持足细胞肌动蛋白细胞骨架。 例如，临床前蛋白尿小鼠模型的功能丧失研究表明，KLF15是 预防足细胞损伤和糖皮质激素(GCs)的有益益处所需的最常见的 对原发性肾小球疾病的治疗，是由KLF15介导的。此外，人类对GCs的反应性 原发性肾小球疾病与足细胞特异性表达KLF15有关。此外，诱导 足细胞中人KLF15可减轻蛋白尿小鼠模型的肾损伤，无不良反应 胃肠道肿瘤后遗症。总之，这些关于KLF15肾脏保护作用的临床前和临床研究 诱导作用促使我们寻找治疗肾脏疾病的新型小分子KLF15激动剂。 我们最初生成并进行了基于细胞的高通量筛选(HTS)，以筛选 诱导KLF15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KLF15所需的最佳治疗窗口内，具有一半最大有效浓度(EC50) 活动。基于EC50和可药性，我们进行了构效关系(SAR)研究 化合物K-7，并产生16个铅类似物，其中BT501、BT502、BT503、BT514和BT412诱导 KLF15启动子活性在细胞应激或不应激的情况下。我们还进行了K-7的初步药代动力学研究。 研究还表明，经K-7和铅类似物处理的人足细胞可减轻足细胞损伤 在细胞应力的设置上。此外，K-7处理的人足细胞的RNA-SEQ显示抑制途径 IL-17RA介导的肌动蛋白细胞骨架失稳，从而为利用 优化KLF15激动剂选择性的方法。最后，我们观察到K-7减轻了蛋白尿和 修复了临床前蛋白尿小鼠模型中的足细胞标志物。基于这些令人信服的初步 数据和先前研究的强大科学严谨性，我们假设领先的KLF15激动剂在 临床前研究将成为蛋白尿肾脏疾病的关键治疗方法。我们建议测试我们的 假说：(1)改善先导KLF15激动剂的药效学和药代动力学性质，(2) 利用机械方法优化KLF15激动剂的选择性，以及(3)测试治疗作用 铅KLF15激动剂在临床前蛋白尿模型中减轻和/或逆转肾脏损伤的作用。 这项提议将通过制定一个综合框架来解决目前该领域的一个空白，以优化 领导新的KLF15激动剂并在临床前蛋白尿模型中测试它们的治疗作用。长期目标 我们项目的重点是确定最佳的KLF15激动剂，该激动剂可以推进用于IND治疗的研究 原发性肾小球疾病。确定治疗蛋白尿疾病的新靶点具有重要意义 考虑到美国退伍军人中CKD的沉重负担，退伍军人管理局对此感兴趣。此外，治疗策略 减少长期使用GC将对与以下疾病相关的并发症产生巨大影响 美国退伍军人的GCS。最后，诱导KLF15的治疗作用可能超越肾脏疾病，因为 其他实验室已经证明了KLF15在心肌肥厚中的有益作用， 神经退行性疾病和脂肪生成。

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.