Novel Drug Discovery for AD Targeting Ryanodine Calcium Channels

 描述（由申请人提供）：目前，没有有效的策略或治疗方法来保护AD患者的认知功能。最近一系列令人失望的临床试验突出了探索替代途径的必要性。能够以不同于以往方法的方式保护认知功能并预防疾病进展的新型化合物可以提供新的治疗机会。为此，我们正在开发和测试小分子化合物，设计为ryanodine受体（RyR）的变构调节剂，ER膜上发现的大电导钙通道，作为早期AD或MCI患者临床测试的候选药物。在人类AD患者和AD小鼠模型中，RyR 2表达增加先于淀粉样蛋白沉积、tau组织病理学、神经元损失和认知障碍。在AD小鼠模型中，增加的RyR诱发的钙释放在树突棘和突触区室中最大，并且有助于突触病理学和功能障碍、增加的淀粉样蛋白和tau病理学、破坏的记忆功能和其他AD定义特征。我们和其他人最近证明，用丹曲林（一种RyR通道稳定剂）治疗AD小鼠，产生了令人兴奋的治疗效果。尽管我们的治疗方案不同，但一致的结果表明钙信号传导正常化（Chakroborty等人，2012 a; Oule等人，2012; Stutzmann等人，2006）、正常突触传递和可塑性表达（Chakroborty等人，2012 a），恢复突触结构和完整性（Briggs等人，2014）、降低的A β水平（Chakroborty等人，2012 a; Oule等人，2012; Peng等人，2012），恢复RyR同种型水平（Chakroborty等人，2012 a; Oule等人，2012），以及改进的存储器测试性能（Oule等人，2012; Peng等人，2012; Stutzmann实验室，未发表的数据）。这些数据支持稳定RyR功能的强有力的案例，重点是RyR 2，作为AD的新治疗策略。本研究的目的是设计，测试和优化化合物，将作为RyR通道负变构调节剂，用于抑制过度的钙释放，同时保持生理功能。中心假设是小分子RyR稳定剂的开发和优化将产生用于早期AD和MCI患者的临床测试的治疗线索，并且通过保持钙稳态和突触功能，将保护认知能力。这将实现以下目标：1。化合物开发和药物化学优化。这将在小鼠中使用迭代药物化学程序和生物活性试验。2. AD小鼠细胞培养系统和神经元的快速筛选试验。初始筛选将使用96孔板中培养的N2 A细胞中RyR诱发的钙信号的自动荧光检测，然后在对照和AD小鼠的原代神经元中进行筛选。3.小鼠模型中的体内验证。在AD和对照小鼠中进行亚慢性治疗，然后进行生理和生物化学测定，以确定和最终确定最佳化合物。对公共卫生的重要性是有效和新颖的AD治疗的可用性。

 DESCRIPTION (provided by applicant): Currently, there are no effective strategies or treatments to preserve cognitive function in AD patients. The recent series of disappointing clinical trials highlight the need to explore alternative pathways. Novel compounds that can preserve cognitive function and prevent disease progression in a manner distinct from previous approaches could provide new therapeutic opportunities. To this end, we are developing and testing small molecule compounds designed as allosteric modulators of the ryanodine receptor (RyR), a large conductance calcium channel found on the ER membrane, as candidates for clinical testing in early AD or MCI patients. In both human AD patients and AD mouse models, increased RyR2 expression precedes the amyloid deposition, tau histopathology, neuronal loss, and cognitive impairments. In AD mouse models, increased RyR-evoked calcium release is greatest in dendritic spines and synaptic compartments, and contributes to synaptic pathology and dysfunction, increased amyloid and tau pathology, disrupted memory function, and other AD-defining features. We and others have recently demonstrated that treating AD mice with dantrolene, a RyR channel stabilizer, resulted in exciting therapeutic effects. Although our treatment regimens differed, the consistent results demonstrate normalized calcium signaling (Chakroborty et al., 2012a; Oule et al., 2012; Stutzmann et al., 2006), normal synaptic transmission and plasticity expression (Chakroborty et al., 2012a), restored synaptic structure and integrity (Briggs et al., 2014), reduced A levels (Chakroborty et al., 2012a; Oule et al., 2012; Peng et al., 2012), restored RyR isoform levels (Chakroborty et al., 2012a; Oule et al., 2012), and improved performance on memory tests (Oule et al., 2012; Peng et al., 2012; Stutzmann lab, unpublished data). These data support a strong case for stabilizing RyR function, with a focus on RyR2, as a novel therapeutic strategy for AD. The objective of this study is to design, test, and optimize compounds that will function as RyR channel negative allosteric modulators, serving to suppress excessive calcium release while maintaining physiological functions. The central hypothesis is that development and optimization of small molecule RyR stabilizers will generate therapeutic leads for clinical testing in early AD and MCI patients, and through the preservation of calcium homeostasis and synaptic function, will protect cognitive abilities. This will be accomplished with the following Aims: 1. Compound development and medicinal chemistry optimization. This will use iterative medicinal chemistry procedures and bioactivity assays in mice. 2. Rapid screening assay in cell culture systems and neurons from AD mice. Initial screening will use automated fluorometric testing of RyR-evoked calcium signals in cultured N2A cells in 96-well plates, followed by screening in primary neurons from control and AD mice. 3. In vivo verification in mouse models. Sub chronic treatment in AD and control mice, followed by physiological and biochemical assays, will then be used to identify and finalize the optimal compounds. The significance to public health is the availability of an effective and novel treatment for AD.