Alzheimer's Disease: simultaneous modulation of NMDAR, BACE1, APP and Fyn activity by palmitoylation-targeting CIRC825

阿尔茨海默病：通过棕榈酰化靶向 CIRC825 同时调节 NMDAR、BACE1、APP 和 Fyn 活性

• 批准号: 10257994
• 负责人: Devin Thomas Wiley
• 金额: $ 50.73万
• 依托单位: CIRCUMVENT PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10257994
• 关键词: 3-Dimensional; Action Potentials; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease therapeutic; Amyloid beta-42; Biochemical Pathway; Biological Assay; Biology; Brain Diseases; CLN1 gene; Calcium; Cell Death; Cell Survival; Cells; Cerebrum; Clinical; Clinical Trials; Cognition; Cognitive deficits; DLG4 gene; Data; Diffusion; Disease; Disease Progression; Doctor of Philosophy; Dose; Drug Targeting; Electroencephalography; Electrophysiology (science); Epilepsy; Event; Fatty acid glycerol esters; Feasibility Studies; Frequencies; Generations; Glutamates; Goals; Grant; Human; Hydroxylamine; Image; Immunohistochemistry; Investigation; J20 mouse; Location; Marshal; Memantine; Modeling; Motor; Mus; Mutation; Namenda; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Neurotransmitter Receptor; Organoids; Palmitates; Pathogenesis; Pathogenicity; Peptides; Performance; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Phosphotransferases; Physiological; Post-Translational Protein Processing; Program Development; Proteins; Psychological Transfer; Reporting; Research Personnel; Seizures; Senile Plaques; Series; Sheep; Synapses; Testing; Therapeutic; Transgenic Mice; Work; beta-site APP cleaving enzyme 1; brain cell; clinical translation; cognitive function; cytotoxicity; drug development; druggable target; excitotoxicity; experimental study; familial Alzheimer disease; improved; induced pluripotent stem cell; motor deficit; mouse model; multi-electrode arrays; new therapeutic target; novel; novel therapeutics; overexpression; palmitoylation; patch clamp; presenilin-1; programs; response; small molecule; tau aggregation; virtual; water maze

PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) has predominantly been characterized by amyloid plaques and neurofibrillary tau tangles, and soluble oligomers of these peptides are thought to be toxic; hence, targeting these histopathological hallmarks has led to a long series of clinical trials, but to date they have all failed. Intriguingly, aberrant palmitoylation of multiple proteins has been reported in AD. Accordingly, we seek here to simultaneously modulate the location and activity of multiple AD-associated proteins (neurotransmitter receptor NMDAR, amyloid plaque-associated BACE1 and APP, and neurofibrillary tangle-associated Fyn kinase) by altering their shared post-translational modification, palmitoylation. To this effort, we will transfer learnings from a drug development program of a small molecule that removes palmitate from proteins, N-(tert-butyl)-hydroxylamine (NtBuHA, called CIRC825) in the well-defined neurodegenerative disease that is associated with over- palmitoylation, CLN1 Batten (CLN1). Of great importance to this AD-related grant, extrasynaptic (e)NMDARs containing predominantly GluN2B subunits have been implicated to be overactivated in the pathogenesis of AD. In a feasibility study demonstrating our approach will work in the context of human AD, our preliminary findings have recently shown that CIRC825 treatment virtually eliminates the aberrant hyperexcitable phenotype in human AD hiPSC cerebrocortical neurons compared to WT. Additionally as feasibility data, we investigated CIRC825 in CHOAPP cells (which overexpress APP), and found it lowered palmitoylation of APP, and it decreased Aβ42 generation (the pathogenic form) without decreasing soluble Aβ40. The aims of this Phase 1 work are focused on expanding our understanding the pathomolecular importance of the palmitoylation of APP, BACE1, Fyn, and NMDAR in both Cln1-/- mouse neurons and in human (h)iPSC-derived 2D cortical neuronal cultures and 3D cerebral organoids that have familial AD mutations. Human iPSCs to be used include wPSEN1 M146V/WT and APPswe/WT hiPSC lines as well as the PSEN1 ΔE9/WT hiPSC line (with WT isogenic controls). We will modulate palmitoylation in these models by treating them with CIRC825. Finally, in a Cln1-/- mouse model, we will compare physiologic benefits of CIRC825 to those of memantine. Specifically, in aim 1 we will evaluate CIRC825 in cultured CLN1-/- neurons and (h)iPSC-derived AD neurons for 1) effects on decreasing palmitoylation of APP, BACE1, Fyn, and NMDAR; 2) suppression of extra-synaptic dendritic Ca2+ influxes by calcium imaging, and 3) mitigation of glutamate-induced cell death in an excitotoxicity assay. In aim 2 we will test concentration-dependent physiologic effects of CIRC825 on (h)iPSC-derived 3D cerebral organoids (with familial AD mutations and WT controls), using multiple assays including patch-clamp electrophysiology, calcium imaging, multi-electrode array experiments, and immunohistochemistry. In aim 3 we will investigate the dose- dependent response of CIRC825 and memantine in CLN1-/- mice, assessing for motor deficits by a rotarod analysis, cognitive deficits by a water-maze and Y-maze assessment, and control of seizures by EEG. On completion of this work, we aim to demonstrate with greater confidence that palmitoylation is a druggable target in AD, which, to our knowledge, is a novel pharmacologic approach in AD. Further, as CIRC825 is being investigated in IND-enabling studies, we aim to develop a data package on CIRC825 that justifies more rigorous investigations of its efficacy in AD models, with the ultimate goal of clinical translation of CIRC825 (or other hydroxylamine derivatives) in AD. Results of this work will guide a Phase II program that is focused on a more targeted dose range assesment in Cln1-/- sheep and investigations in a broader set of AD models, including in hAPP-J20 and 5XfAD transgenic mice.

项目总结/摘要 阿尔茨海默病（AD）的主要特征是淀粉样斑块和神经胶质tau蛋白 缠结和这些肽的可溶性低聚物被认为是有毒的;因此，靶向这些组织病理学的 hallmarks导致了一系列的临床试验，但迄今为止，它们都失败了。有趣的，异常的 在AD中已经报道了多种蛋白质的棕榈酰化。因此，我们在此寻求同时 调节多种AD相关蛋白（神经递质受体NMDAR， 淀粉样斑块相关的BACE 1和APP，以及神经元缠结相关的Fyn激酶）。 共有的翻译后修饰棕榈酰化。为了实现这一目标，我们将从一种药物中 一种能从蛋白质中去除棕榈酸的小分子N-叔丁基羟胺的开发计划 （NtBuHA，称为CIRC 825）在定义明确的神经退行性疾病中的作用， 棕榈酰化，CLN 1 Batten（CLN 1）。对于这种AD相关补助金非常重要的是，突触外（e）NMDAR 主要含有GluN 2B亚基的蛋白质在AD的发病机制中被过度激活。 在一项证明我们的方法将在人类AD背景下工作的可行性研究中，我们的初步发现 最近表明，CIRC 825治疗实际上消除了异常的过度兴奋表型， 与WT相比，人AD hiPSC的皮质神经元。此外，作为可行性数据，我们调查了 CIRC 825在CHOAPP细胞（过度表达APP）中的作用，发现它降低了APP的棕榈酰化， Aβ42生成（致病形式）而不降低可溶性Aβ40。第一阶段工作的目标是 专注于扩大我们对APP，BACE 1， Fyn和NMDAR在Cln 1-/-小鼠神经元和人（h）iPSC衍生的2D皮质神经元培养物中的表达 和具有家族性AD突变的3D脑类器官。待使用的人iPSC包括wPSEN 1 M146 V/WT和APPswe/WT hiPSC系以及PSEN 1 ΔE9/WT hiPSC系（具有WT同基因对照）。 我们将通过用CIRC 825处理这些模型来调节棕榈酰化。最后，在Cln 1-/-小鼠中， 模型，我们将比较CIRC 825的生理效益美金刚。具体而言，在目标1中，我们将 评估培养的CLN 1-/-神经元和（h）iPSC衍生的AD神经元中的CIRC 825的1）降低AD神经元的作用 APP、BACE 1、Fyn和NMDAR的棕榈酰化; 2）通过抑制突触外树突状Ca 2+内流， 钙成像，和3）在兴奋性毒性测定中减轻谷氨酸诱导的细胞死亡。在目标2中， 测试CIRC 825对（h）iPSC衍生的3D脑类器官（具有 家族性AD突变和WT对照），使用多种测定，包括膜片钳电生理学、钙离子通道、钙离子通道和钙离子通道。 成像、多电极阵列实验和免疫组织化学。在目标3中，我们将研究剂量- CLN 1-/-小鼠中的CIRC 825和美金刚的依赖性反应，通过旋转棒评估运动缺陷 分析，通过水迷宫和Y迷宫评估认知缺陷，以及通过EEG控制癫痫发作。对 完成这项工作后，我们的目标是更有信心地证明棕榈酰化是一个可药用的目标 据我们所知，这是一种治疗AD的新药理学方法。此外，由于CIRC 825正在 在IND使能研究中进行了调查，我们的目标是开发一个关于CIRC 825的数据包， 研究其在AD模型中的功效，最终目标是临床转化CIRC 825（或其他 羟胺衍生物）。这项工作的结果将指导第二阶段的计划，重点是一个更 Cln 1-/-绵羊中的靶向剂量范围评估和更广泛AD模型组中的研究，包括 hAPP-J20和5XfAD转基因小鼠。