Novel small molecules targeting brain phospholipid dysregulation in AD

阿尔茨海默病（AD）是最常见的老年性神经退行性疾病，八分之一的老年人 被诊断为AD的美国人它也是退伍军人中最常见的痴呆症类型 事务（VA）医疗系统，和退伍军人发病率和死亡率的主要原因之一。的 退伍军人事务部估计，60万退伍军人患有严重的AD和其他形式的痴呆症， 2000年，由于老年退伍军人的比例不断增加， 以及患有创伤性脑损伤（TBI）和/或脑损伤后的退伍军人中痴呆症的患病率增加。 创伤性应激障碍（PTSD）。因此，AD研究对退伍军人尤为重要。 目前没有治疗方法可以减缓或停止AD。非常需要确定更多的 有效治疗AD，这是VA RR & D研究方向的优先事项之一。 Synaptojanin 1（synj1）是细胞内主要的磷酸肌醇二磷酸酶[PIP2降解酶]， 大脑和突触，最近与AD有关。更重要的是，我们已经证明了synj1 调节A β的溶酶体清除，synj1表达增加与ApoE4诱导的 磷脂失调和认知缺陷。下调synj1可促进A β清除， tau过度磷酸化并改善ApoE4致病作用。随后，synj1的还原 减轻AD小鼠模型中AD相关的病理变化和行为缺陷。这些发现 提示synj1减少对AD具有潜在的治疗益处。我们初步筛选了89个最佳人选 一个化合物库（约3，600个小分子）具有降低synj1蛋白水平的潜力， 连接图"，确定了一种FDA批准的药物尼莫地平，在野生型和非野生型小鼠中均有降低synj 1和A β的作用。 型和ApoE4神经元培养物。尼莫地平进一步给药一个月，能够减少 synj1和A β的脑含量，以及改善AD转基因小鼠模型的认知功能， ApoE4 KI小鼠模型。然而，尼莫地平长期给药不能降低脑A β 42水平 （特别是不溶性部分）或改善认知功能。我们的数据表明， 尼莫地平抑制synj1表达与其对钙通道的抑制无关 活动然后，我们设计并合成了第一代尼莫地平结构类似物， 化学，以降低其钙通道活性，并确定了一种新的化合物，SynaptoCpd#9， 减弱钙通道抑制，并增加对synj1和A β 42的效力， 尼莫地平在体外和体内的作用。在APPSwe/PS1/ApoE 9和ApoE 4小鼠中口服施用SynaptoCpd#9 持续3 - 6个月，改善认知功能，减少AD相关病变（特别是不溶性A β 42）。 经处理的ApoE4神经元的RNA测序和qPCR研究鉴定了三个参与ApoE4神经元表达的候选基因。 尼莫地平或SynaptoCpd#9介导的作用。我们认为，继续探索尼莫地平的SAR 衍生物及其作用机制的进一步剖析将为AD提供新的见解 发病机制，并可能导致识别新的目标，为AD治疗。 在本申请中，我们提出改进尼莫地平衍生物的结构修饰，以增加其 在AD小鼠模型中降低synj1和改善认知功能的效力（目的1：药物化学 修改、体外测试漏斗测定和概念动物研究的体内证明）。在平行研究中，我们 建议剖析这些新化合物的作用机制（目标2：机制研究），这将 为AD的发病机制和治疗策略提供了新的见解。 从我们拟议的研究中获得的信息将导致开发新的有效的AD疗法， 通过机制研究更好地了解AD发病机制。因此，我们的研究将直接 使退伍军人受益，并提高VA医疗保健系统内提供的服务质量。

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease of aging, with one in eight older Americans diagnosed with AD. It is also the most frequently diagnosed type of dementia within the Veterans Affairs (VA) Medical System, and one of the major causes of morbidity and mortality among veterans. The Department of VA estimates that 600,000 veterans suffered from severe AD and other forms of dementia in 2000, and this number is increased significantly today, because of the increasing proportion of older veterans and the increased prevalence of dementia in veterans that suffer from traumatic brain injury (TBI) and/or post- traumatic stress disorder (PTSD). Therefore, AD research studies are particularly important to veterans. Currently no treatment is available to slow or stop AD. There is a great need for identification of more efficacious therapies for AD, which is among the priorities of VA RR&D research directions. Synaptojanin 1 (synj1), the main phosphoinositol biphosphate phosphatase [PIP2 degrading enzyme] in the brain and synapses, has been recently linked to AD. More importantly, we have demonstrated that synj1 regulates lysosomal clearance of Aβ and that the increased synj1 expression links to ApoE4-induced phospholipid dysregulation and cognitive deficits. Down-regulation of synj1 promotes Aβ clearance, reduces tau hyper-phosphorylation and ameliorates ApoE4 pathogenic effects. Subsequently, reduction of synj1 attenuates AD-related pathological changes and behavioral deficits in AD mouse models. These findings suggest that reduction of synj1 has potential therapeutic benefits for AD. Our initial screening of 89 top hits out of a library of compounds (~3,600 small molecules) with the potential to reduce synj1 protein levels using “The Connectivity Map”, identified a FDA-approved drug nimodpine with synj1- and Aβ-lowering effects in both wild- type and ApoE4 neuronal cultures. Further administration of nimodipine for one month, is capable of reducing brain content of synj1 and Aβ, as well as improving cognitive functions in an AD transgenic mouse model and an ApoE4 KI mouse model. However, chronic administration of nimodipine failed to reduce brain Aβ42 levels (particularly insoluble fractions), or to improve cognitive function. Our data suggest that the effects of nimodipine on reduction of synj1 expression are independent of its inhibitory effects on calcium channel activities. We then designed and synthesized first-generation nimodipine structural analogs using medicinal chemistry to reduce its calcium channel activity, and identified a novel compound, SynaptoCpd#9, with attenuated inhibition of calcium channels and increased potency against synj1 and Aβ42 compared to nimodipine both in vitro and in vivo. Oral administration of SynaptoCpd#9 in APPSwe/PS1E9 and ApoE4 mice for 3-6 months improved cognitive function and reduced AD-related pathologies (insoluble Aβ42 particularly). RNA-sequencing and qPCR studies of treated ApoE4 neurons identified three candidate genes involved in nimodipine- or SynaptoCpd#9-mediated effects. We posit that continued exploration of SAR of nimodipine derivatives and further dissection of their mechanisms of actions will provide new insights regarding AD pathogenesis and may lead to identification of novel targets for AD therapies. In this application, we propose to refine structural modifications of nimodipine derivatives to increase their potency at lowering synj1 and improving cognitive function in AD mouse models (aim 1: medicinal chemistry modifications, in vitro test funnel assays and in vivo proof of concept animal studies). In parallel studies, we propose to dissect mechanisms of actions of these novel compounds (aim 2: mechanistic studies), which will provide new insights regarding mechanisms of AD pathogenesis and advance therapeutic strategies for AD. Information obtained from our proposed studies will lead to development of novel potent AD therapies, and better understanding of AD pathogenesis through mechanistic investigation. Therefore, our studies will directly benefit Veterans, and improve the quality of service provided within the VA health care system.