Drug Discovery Pipeline Targeting Pathologically Leaky Calcium Release Channels in Age-Related Indications

项目摘要 Phoonic Pharma LLC(PP)提出了一项第一阶段SBIR项目，以启动高通量筛选(HTS) 病理性Ryanodine受体(RyR)钙(Ca)小分子变构抑制剂的发现 释放渠道，以阻止或逆转与年龄相关的神经退化，专注于阿尔茨海默病 (Ad)。在阿尔茨海默病中，细胞内钙调节失调，引发了一种病理生理恶性循环。RYR负责 触发细胞内存储细胞器的钙释放(肌浆网；内质网 -ER)。由此产生的钙信号对肌肉和非肌肉细胞中的许多细胞过程都是必不可少的。 RyR功能失调，导致静息ER/SR的钙泄漏状态，已被认为是一个关键 导致与年龄相关的疾病，尤其是阿尔茨海默病。在这个提议中，我们瞄准了RyR2，即占主导地位的大脑 亚型，它已被明确地确定为AD发病机制和治疗的靶点。我们有 将我们在UMN的学术研究转化为针对RyR2的基于荧光的HTS结构分析 (美国专利10,281,476)。FRET检测使用荧光标记的RyR2调节剂、钙调蛋白(CaM)和 FKBP12.6(FKBP)，用于监测RyR2-CaM结合。使用小的试点屏幕，我们已经演示了 我们的FRET读数和静息RyR2活性(钙泄漏)的复合效应之间存在负相关。这 建立了HTS平台，以确定具有治疗潜力的变构调节剂来缓解RyR2病例 漏水。在我们在密歇根大学的学术工作中，我们获得了一笔R01拨款，用于将这项技术应用于研究RyR2 心脏的功能和治疗；PP已经获得了这项UMN专利的独家商业许可。在这 项目，我们将应用这项技术进行治疗发现，以纠正细胞内钙信号。 阿尔茨海默氏症。在第一阶段，我们将建立一个筛选漏斗，用于(A)大规模高温超导运动 以及(B)通过结构-活性关系(SAR)研究随后的铅开发。在第二阶段，我们会 扩展SAR，并开始使用AD模型进行基于细胞和动物的研究。实现阶段性目标 我，PP(1)已经展示了将此FRET系统应用于RyR2目标HTS的经验，(2)将使用其 专有的高精度、高通量的Flt平板阅读器(Flt-PR)，(3)具有功能验证的命中 该HTS平台产生的RyR2，以及(4)在心脏中发现了RyR2的泄漏抑制剂。第一阶段的具体目标是： 目标1-50,000种化合物CNS选择文库的初级HTS，以鉴定纠正 漏水的RyR状态。目标2-确定来自目标1的命中的功能效果。我们已经组装了一个 杰出的跨学科团队，包括来自几所大学的学术专家(药物化学家 UMN的Aldrich；Rosalind Franklin大学的RyR2 AD专家Stutzmann，人类RyR2专家Zima 洛约拉大学芝加哥分校，补充了光子团队在AD目标HTS方面的专业知识。这些 合作，以及我们与一家大型制药公司成功合作的记录，结合在一起 确保该项目未来的成功商业化。

Project Summary Photonic Pharma LLC (PP) proposes a Phase I SBIR project to launch high-throughput screening (HTS) for discovery of small-molecule allosteric inhibitors of pathologically leaky ryanodine receptor (RyR) calcium (Ca) release channels, to arrest or reverse age-related neurodegeneration, focused on Alzheimer’s disease (AD). In AD, dysregulated intracellular Ca fuels a pathophysiological vicious cycle. RyRs are responsible for triggered Ca release from intracellular storage organelles (sarcoplasmic reticulum – SR; endoplasmic reticulum – ER). The resulting Ca signaling is essential for many cellular processes in muscle and non-muscle cells. Dysregulated RyR function, producing a Ca-leaky state of the resting ER/SR, has been identified as a key contributor to age-related pathologies, particularly AD. In this proposal, we target RyR2, the predominant brain isoform, which has been clearly identified as a target for AD pathogenesis and therapy. We have translated our academic research at UMN toward fluorescence-based HTS structural assays targeting RyR2 (US patent 10,281,476). This FRET assay uses fluorescently labeled RyR2 modulators, calmodulin (CaM) and FKBP12.6 (FKBP), to monitor RyR2-CaM binding. Using small pilot screens, we have demonstrated an inverse correlation between compound effects on our FRET readout and resting RyR2 activity (Ca leak). This established an HTS platform to identify allosteric regulators with therapeutic potential to alleviate RyR2 Ca leak. In our academic work at UMN, we have a funded R01 grant to apply this technology to studying RyR2 function and therapy in the heart; PP has obtained an exclusive commercial license for this UMN patent. In this project, we will apply this technology for therapeutic discovery to correct intracellular Ca signaling in Alzheimer’s disease. In Phase I, we will establish a screening funnel for (a) a large-scale HTS campaign and (b) subsequent lead development via structure-activity relationship (SAR) studies. In Phase II, we will expand SAR, and begin cell-based and animal studies using models of AD. To achieve the objectives in Phase I, PP (1) has demonstrated experience applying this FRET system for RyR2-targeted HTS, (2) will use its proprietary high-precision, high-throughput FLT plate reader (FLT-PR), (3) has functionally validated Hits yielded by this HTS platform, and (4) has identified leak inhibitors of RyR2 in heart. The Phase I Specific Aims: Aim 1 – Primary HTS of a 50,000-compound CNS-selected library, to identify compounds that correct the leaky RyR state. Aim 2 – Determine functional effects of Hits from Aim 1. We have assembled an outstanding interdisciplinary team, including academic experts from several universities (medicinal chemist Aldrich at UMN; RyR2 AD expert Stutzmann at Rosalind Franklin University, human RyR2 expert Zima at Loyola University Chicago, complementing the Photonic team’s expertise on AD-targeted HTS. These collaborations, and our track record of successful collaboration with a major drug company, combine to ensure the successful future commercialization of this project.