Myosin Activation as a Novel Therapeutic Strategy for Polycystic Kidney Disease

摘要 常染色体显性遗传性多囊肾病(ADPKD)在全球每400-1000人中就有1人患病， 导致肾脏、肝脏和其他器官不断产生充满液体的包囊，导致器官衰竭。 PKD是由多囊蛋白多囊蛋白-1(PC1)或多囊-2(PC2)突变引起的。一目了然 多囊蛋白功能障碍是如何从机制上导致囊性病变的，目前尚不清楚，部分原因是 缺乏能够准确反映疾病表型的包囊生成模型系统。干细胞来源的人肾 有机化合物以一种特定的方式模拟PKD囊变，并有可能加速药物发现和 PKD的发展。 使用这些PKD有机化合物，进行药物筛选，确定肌球蛋白抑制剂Blebbistatin是一种 有效的囊性激动剂。随后，我发现EMD，一种肌球蛋白激活剂，在 在疾病的早期和晚期用PKD器官类物质治疗PKD的囊变。基于初步的 来自文献的数据和线索，我假设PC1和PC2正向调节非肌肉肌球蛋白 肾小管上皮细胞内的收缩能力，以防止囊变。 利用独特的肾脏有机化合物体外系统，我建议分析肌球蛋白调节剂化合物和 有机体中的非肌肉肌球蛋白动力学，以精确定位控制囊肿胀大的肌球蛋白结构域 通知临床前药物开发。第二，我的目标是优化体内给药和治疗策略 小鼠PKD模型中的EMD。最后，我将利用PC1在结构上类似于非典型 黏附G蛋白偶联受体分析生化激活PC1能否刺激类有机物 收缩将多囊蛋白功能与肌球蛋白控制联系起来。总之，这些实验将建立肌球蛋白 激活作为治疗PKD的策略和确定PC1作为肌球蛋白调节因子的机制 伸缩性。这项工作的目标是通过利用最好的模型系统来加速PKD的药物发现 代表疾病，以及告知多囊藻毒素在囊变中的作用的机制研究。

ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) affects 1 in every 400-1000 people worldwide, causing progressive fluid-filled cyst production in the kidneys, liver, and other organs, resulting in organ failure. PKD is caused by mutations in the polycystin proteins polycystin-1 (PC1) or polycystin-2 (PC2). A clear understanding of how polycystin dysfunction leads to cysts mechanistically is still elusive, due, in part, to the lack of cyst producing model systems that accurately phenocopy the disease. Stem cell derived human kidney organoids model PKD cystogenesis in a specific way and have potential to accelerate drug discovery and development for PKD. Using these PKD organoids, a drug screen was performed identifying blebbistatin, a myosin inhibitor, as a potent cyst agonist. Subsequently, I discovered that EMD, a myosin activator, showed pre-clinical promise in treating PKD cystogenesis in PKD organoids at both early and late stages of disease. Based upon preliminary data and clues from the literature, I hypothesize that PC1 and PC2 positively regulate non-muscle myosin contractility within the tubular epithelium to prevent cystogenesis. Utilizing the unique kidney organoid in vitro system, I propose to analyze myosin modulator compounds and non-muscle myosin dynamics in organoids to pinpoint the myosin domain that governs cyst expansion to inform pre-clinical drug development. Second, I aim to optimize in vivo delivery and treatment strategies of EMD in a mouse model of PKD. Finally, I will utilize the insight that PC1 structurally resembles an atypical adhesion G-protein coupled receptor to analyze whether biochemically activating PC1 can stimulate organoid contraction to link polycystin function to myosin control. Together, these experiments will establish myosin activation as a therapeutic strategy to treat PKD and determine mechanisms of PC1 as a regulator of myosin contractility. The goal of this work is to expedite drug discovery for PKD by utilizing model systems that best represent the disease, and mechanistic studies that inform polycystin function in cystogenesis.