Evaluating the p-Tau inhibition and neuroprotective effects of sAPPalpha using brain permeable small molecules

使用脑通透性小分子评估 sAPPalpha 的 p-Tau 抑制和神经保护作用

• 批准号: 10522638
• 负责人: Varghese John
• 金额: $ 39万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522638
• 关键词: 3xTg-AD mouse; ADME Study; Acute; Aducanumab; Affect; Affinity; Agonist; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Alzheimer' s disease therapy; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Antibodies; Artificial Membranes; Behavioral; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Availability; Brain; Cause of Death; Cell Membrane Permeability; Cells; Chemicals; Chemistry; Chronic; Cognition; Cognitive; Corticotropin-Releasing Hormone; Country; Data; Development; Dose; Drug Kinetics; Enhancers; Evaluation; Extracellular Domain; FDA approved; Goals; HTR3A gene; Hand; Hippocampus (Brain); Histopathology; Human; Impaired cognition; In Vitro; Induced pluripotent stem cell derived neurons; Injections; J20 mouse; Ligands; Membrane; Memory; Mus; Muscarinic M1 Receptor; Muscarinic M3 Receptor; Neurites; Neurofibrillary Tangles; Neurons; Oral; Outcome; Pathology; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphorylation Site; Phosphotransferases; Postoperative Nausea and Vomiting; Property; Reporting; Running; Safety; Serotonin Antagonists; Site; Solubility; Structure-Activity Relationship; Synapses; Tauopathies; Testing; Tropisetron; Vertebral column; acetylcholine receptor agonist; aged; alpha-bungarotoxin receptor; analog; antagonist; apolipoprotein E-4; base; biological adaptation to stress; cognitive benefits; combinatorial; density; design; efficacy study; improved; in vivo; in vivo evaluation; induced pluripotent stem cell; lead candidate; mouse model; neuroblastoma cell; neurofibrillary tangle formation; neuroprotection; new therapeutic target; novel therapeutic intervention; novel therapeutics; phosphoproteomics; presenilin-1; receptor; receptor binding; response; screening; serotonin receptor; small molecule; tau Proteins; tau aggregation; tau phosphorylation; tau-1; therapeutic candidate; therapeutically effective

PROJECT SUMMARY/ABSTRACT We previously reported (2) on the soluble amyloid precursor protein alpha (sAPPα)-enhancing effects of tropisetron (F03). Highly brain-permeable F03 is approved in 49 countries for the treatment of post-operative nausea and vomiting (PONV) and is a multifunctional ligand: it is a potent 5-HT3 serotonin receptor (5-HT3R) antagonist (Ki ~ 3 nM), a partial α7 nicotinic acetylcholine receptor (α7nAChR) agonist (Ki ~ 450 nM), and binds to the extracellular domain of amyloid precursor protein (eAPP). In our studies, we found F03 increases sAPPα and also significantly decreases the phospho-tau (p-tau)/total tau (t-tau) ratio in two Alzheimer's disease (AD) mouse models. We show that F03 can also reduce corticotropin-releasing factor (CRF) induced p-tau/tau increase in vitro. Hyperphosphorylation of tau leads to toxic tau oligomers and ultimately formation of neurofibrillary tangles (NFTs) in AD brain and is closely correlated with cognitive decline (34); thus, decreasing tau phosphorylation is a critical target for new therapeutic approaches for AD and tauopathies. Based on reports that sAPPα reduces the p-tau/t-tau ratio through suppression of activity of the kinase GSK3β (9), we plan to assess the ability of our sAPPα enhancers to reduce the p-tau/t-tau ratio in vitro and in vivo. A key goal of the project is to identify optimized small molecule sAPPα-enhancing, p-tau/t-tau lowering compounds that improve cognition in murine AD models, for further development as a novel therapy for AD. An additional goal would be to elucidate the underlying mechanism of action (MOA) of sAPPα enhancement and related reduction in the p- tau/t-tau ratio. In Aim 1, we would test F03 and analogs we have in-hand as well as new analogs from Aim 2 in SH-SY5Y cells and 3xTg-AD primary neurons to establish EC50s for sAPPα enhancement and p-tau/t-tau decreases. Prioritized compounds would be evaluated for receptor binding and APP binding. Tertiary testing will be in induced pluripotent stem cell (iPSC)-derived neurons from AD subjects and includes synaptic spine density quantification. In Aim 2, medicinal chemistry would be used to design new chemical entity (NCE) analogs in a iterative fashion with receptor and APP binding along with enhanced drug-like properties and oral brain permeability. In Aim 3, ADME studies on analogs include solubility, microsomal stability, protein binding, parallel artificial membrane permeability assay (PAMPA) analysis, and in vivo pharmacokinetics (PK). Optimal candidates would undergo safety (including hERG) and off-target panel profiling along with analyses of effect on other kinases and of tau phosphorylation sites. In Aim 4 in vivo testing, including acute studies on select candidates to evaluate effect on intracerebroventricular (ICV) delivered CRF induced p-tau/t-tau increases in brain in 3xTg-AD mice. Active candidates would be tested in chronic 4-week efficacy studies in 3xTg-AD and ApoE4-5XFAD AD model mice. Readouts would include behavioral and histopathology analysis, including hippocampal neurite load and neuronal spine density. Biochemical outcomes include p-tau/t-tau ratio, sAPPα and Aβ. Mechanistic studies by phosphoproteomics used for correlations between readouts and cognition.

项目摘要/摘要 我们之前报道了(2)可溶性淀粉样前体蛋白α(sAPPα)对 托烷司琼(F03)。高度脑透性的F03在49个国家被批准用于手术后的治疗 恶心和呕吐(PONV)是一种多功能配体：它是一种有效的5-HT3 5-羟色胺受体(5-HT3R) 拮抗剂(Ki~3 nM)，部分α7烟碱型乙酰胆碱受体(α7nAChR)激动剂(Ki~450 nM)，并结合 与淀粉样前体蛋白(EAPP)的胞外结构域有关。在我们的研究中，我们发现F03会增加sAPPα 还显著降低了两个阿尔茨海默病(AD)患者的磷酸化tau(p-tau)/总tau(t-tau)的比率 老鼠模型。我们发现F03还可以降低促肾上腺皮质激素释放因子(CRF)诱导的p-tau/tau 在体外增加。Tau的过度磷酸化导致有毒的tau低聚物，最终形成 AD大脑中的神经原纤维缠结(NFT)与认知功能下降密切相关(34)；因此，减少 Tau的磷酸化是AD和tauopathy新治疗方法的关键靶点。基于报告 SAPPα通过抑制β的活性来降低p-tau/t-tau比率，我们计划 评估我们的SAPPα增强剂在体外和体内降低p-tau/t-tau比率的能力。的一个关键目标是 项目是寻找优化的小分子sapp-α增强，p-tau/t-tau降低化合物，改善 在小鼠AD模型中的认知，为进一步开发一种治疗AD的新方法奠定基础。另一个目标是 目的：阐明sAPPα增强和相关降低在p-DNA损伤中的作用机制。 Tau/t-tau比率。在目标1中，我们将测试F03和我们手头已有的类似物，以及来自目标2的新类似物 SH-SY5Y细胞和3xTg-AD原代神经元为sAPPα增强和p-tau/t-tau建立EC50 减少。将评估优先化合物的受体结合和APP结合。三级测试将 阿尔茨海默病患者诱导的多能干细胞(IPSC)来源的神经元中的BE，包括突触棘密度 量化。在目标2中，药物化学将被用来设计新的化学实体(NCE)类似物 受体和APP结合的迭代方式以及增强的类药物特性和口服脑 渗透性。在目标3中，ADME对类似物的研究包括溶解度、微粒体稳定性、蛋白质结合、平行 人工膜通透性分析(PAMPA)和体内药代动力学(PK)。最优 候选人将接受安全(包括HERG)和脱离目标的小组分析，以及对 其他激酶和tau磷酸化位点。在AIM 4体内测试，包括对SELECT的急性研究 脑室注射CRF对大鼠脑内p-tau/t-tau升高的影响 3xTg-AD小鼠的脑组织。积极的候选人将接受为期4周的3xTg-AD和 ApoE4-5XFAD模型小鼠。读数将包括行为和组织病理学分析，包括 海马神经元突起负荷和神经元棘密度。生化结果包括p-tau/t-tau比率、SAPPα 还有一辆β。磷酸化蛋白质组学用于读数和认知之间的相关性的机制研究。