Novel Drug Discovery for AD Targeting Ryanodine Calcium Channels

 描述(申请人提供)：目前，尚无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显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和防止疾病进展的新型化合物可能提供新的治疗机会。为此，我们正在开发和测试作为兰尼定受体(RyR)变构调节剂的小分子化合物，RyR是一种发现在ER膜上的大电导钙通道，作为AD或MCI患者早期临床测试的候选药物。在人类AD患者和AD小鼠模型中，RyR2表达增加先于淀粉样蛋白沉积、tau组织病理学、神经元丢失和认知损害。在AD小鼠模型中，RyR诱导的钙释放增加在树突棘和突触间隔最大，并导致突触病理和功能障碍，淀粉样蛋白和tau病理增加，记忆功能障碍，以及其他AD定义特征。我们和其他人最近证明，用丹曲林治疗AD小鼠，一种RyR通道稳定剂，产生了令人兴奋的治疗效果。尽管我们的治疗方案不同，但一致的结果显示钙信号转导正常化(Chakroborty等人，2012a；Oule等人，2012年；Stutzmann等人，2006年)，突触正常传递和可塑性表达(Chakroborty等人，2012a)，恢复突触结构和完整性(Briggs等人，2014年)，降低A水平(Chakroborty等人，2012a；Oule等人，2012年；Peng等人，2012年)，恢复RyR异构体水平(Chakroborty等人，2012a；Oule等人，2012年)，以及记忆测试中的表现改善(Oule等人，2012年；Oule等人，2012年)彭等人，2012；斯图兹曼实验室，未公布的数据)。这些数据支持稳定RyR功能的有力证据，重点是RyR2，作为AD的一种新的治疗策略。本研究的目的是设计、测试和优化作为RyR通道负变构调节剂的化合物，在保持生理功能的同时抑制过量的钙释放。中心假设是，小分子RyR稳定剂的开发和优化将为早期AD和MCI患者的临床测试产生治疗线索，并通过保护钙稳态和突触功能，保护认知能力。这项工作的目标如下：1.化合物开发和药物化学优化。这将在小鼠身上使用迭代的药物化学程序和生物活性分析。2.AD小鼠细胞培养体系和神经元的快速筛选初步筛选将使用96孔板中培养的N2A细胞中RyR诱发的钙信号的自动荧光测试，随后将在对照组和AD小鼠的原代神经元中进行筛选。3.小鼠模型的体内验证。在AD和对照组小鼠中进行亚慢性治疗，然后进行生理和生化分析，然后将用于鉴定和最终确定最佳化合物。对公共卫生的意义在于AD的有效和新的治疗方法的可获得性。

 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.