Targeting Mek/Erk Signaling in the Treatment of Mitral Valve Prolapse

摘要 二尖瓣脱垂（MVP）是最常见的心脏瓣膜疾病之一，每40例患者中就有1例 人类和约70%的小型犬。MVP尚无有效的非手术治疗方法， 由于对其根本原因的不完全理解，治疗努力受到阻碍。最近 我们小组的研究已经描述了非综合征型MVP的遗传基础，并提供了新的见解 转化为疾病的分子过程我们利用这些基因信息来识别 可以为人类提供非手术选择的目标。特别是，我们已经确定了Mek/Erk 途径作为主要的疾病引发途径，Mek/Erk的长期过度活化驱动疾病 进展和严重程度。Mek/Erk途径的激活导致增殖增加、异常增殖和细胞凋亡。 分化和基质金属肽酶（MMP）的产生。因此，我们假设， Mek/Erk活性的药理学阻断将充分阻止疾病途径， 将瓣膜维持在亚临床状态。这一假设将通过筛选新的和FDA- 体外（Aim 1）和体内（Aim 2）批准的丝裂原活化蛋白激酶激酶（MEK 1）抑制剂。要求1 该提案的一项内容涉及使用犬MVP和人二尖瓣体外筛选MEK 1抑制剂 间质细胞，以及使用非活化的MEK 1蛋白进行酶动力学。MEK 1是一种 在Mek/Erk信号通路中有吸引力的治疗靶点。MEK 1抑制剂耐受性良好，FDA- 批准用于黑色素瘤、非小细胞肺癌（NSCLC）和结直肠癌（CRC）;然而， FDA批准和临床试验中的单一MEK 1抑制剂与主要细胞色素相互作用和/或使患者 远离有害的脱靶副作用我们的团队已经鉴定出新型的、高选择性的MEK 1抑制剂， 在计算机模拟中表现出优异的对接分数，并且不抑制主要的细胞色素。因此，在目标1中，我们将测试 假设可以使用计算机辅助药物设计开发上级MEK 1抑制剂。在目标2中， 将评估MEK 1药物治疗在小鼠和犬MVP患者中的功效。在实验1中，我们将 向Dzip 1 S24 R/+小鼠施用新的和FDA批准的MEK 1抑制剂; 表型模仿人类MVP。读数将包括：从鼠标获得的功能测量值 超声心动图;瓣膜体积定量;组织病理学和RNA测序。在实验2中，我们 我计划用40只B1期狗进行双盲、安慰剂对照临床试验来检验我们的假设 每组MVP（安慰剂或曲美替尼）。读数将包括超声心动图、全血细胞计数， 血清生化、NT-proBNP和高敏心肌肌钙蛋白（hs-cTnI）。本研究结果可 作为开发人类MVP患者的第一种非手术治疗的垫脚石。在 除了更好地了解MVP和潜在的治疗方法外，该奖学金还将 为我提供尖端技术的培训，以支持我作为一名医生科学家的发展。

ABSTRACT Mitral valve prolapse (MVP) is one of the most common forms of cardiac valve disease, affecting 1 in 40 humans and ~70% of small breed dogs. There are no effective nonsurgical treatments for MVP and therapeutic efforts have been hindered due to an incomplete understanding of its fundamental causes. Recent studies by our group have described the genetic basis for non-syndromic MVP and have provided new insights into molecular processes that underlie the disease. We have used this genetic information to identify druggable targets that can provide a non-surgical option for humans. In particular, we have identified the Mek/Erk pathway as the major disease initiating pathway and prolonged hyperactivation of Mek/Erk drives disease progression and severity. Activation of the Mek/Erk pathway leads to increased proliferation, aberrant differentiation, and production of matrix metallopeptidases (MMPs). Thus, we hypothesize that pharmacological blockade of Mek/Erk activities will sufficiently arrest the disease pathway and maintain the valves in a sub-clinical condition. This hypothesis will be tested by screening novel and FDA- approved mitogen-activated protein kinase kinase (MEK1) inhibitors in vitro (Aim 1) and in vivo (Aim 2). Aim 1 of this proposal involves an in vitro screen of MEK1 inhibitors using canine MVP and human mitral valve interstitial cells, as well as performing enzyme kinetics using non-activated MEK1 protein. MEK1 is an attractive therapeutic target within the Mek/Erk signaling pathway. MEK1 inhibitors are well-tolerated and FDA- approved for melanoma, non-small cell lung cancer (NSCLC), and colorectal cancer (CRC); however, every single MEK1 inhibitor, both FDA-approved and in clinical trials, interacts with major cytochromes and/or suffers from deleterious off-target side-effects. Our group has identified novel, highly selective, MEK1 inhibitors that exhibit exceptional docking scores in silico and do not inhibit major cytochromes. Thus, in Aim 1 we will test the hypothesis that superior MEK1 inhibitors can be developed using computer-aided drug design. In Aim 2, we will assess the efficacy of MEK1 drug treatment in mouse and canine MVP patients. In experiment 1, we will administer both novel and FDA-approved MEK1 inhibitors to Dzip1S24R/+ mice; a disease model that phenocopies human MVP. Readouts will include: functional measurements obtained from mouse echocardiography; quantification of valve volume; histopathology, and RNA-sequencing. In experiment 2, we plan to test our hypothesis with a double-blinded, placebo-controlled clinical trial with 40 dogs with stage B1 MVP in each arm (placebo or trametinib). Readouts will include echocardiography, complete blood count, serum chemistry, NT-proBNP, and high sensitivity cardiac troponin (hs-cTnI). Results from this study may serve as a stepping stone towards developing the first non-surgical treatment for human patients with MVP. In addition to developing a better understand of MVP and potential therapeutic approaches, this fellowship will provide me with training in cutting-edge techniques that will support my development as a physician scientist.