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.

项目总结/摘要 我们先前报道了（2）可溶性淀粉样前体蛋白α（sAPPα）增强作用， 托烷司琼（F03）。高脑渗透性F03在49个国家获批用于治疗术后 恶心和呕吐（PONV），是一种多功能配体：它是一种有效的5-HT 3血清素受体（5-HT 3R） 拮抗剂（Ki ~ 3 nM），部分α7烟碱乙酰胆碱受体（α 7 nAChR）激动剂（Ki ~ 450 nM），并结合 淀粉样前体蛋白（eAPP）的胞外结构域。在我们的研究中，我们发现F03增加sAPPα， 并且还显著降低了两种阿尔茨海默病（AD）中磷酸化tau（p-tau）/总tau（t-tau）的比率， 小鼠模型。我们发现F03也可以减少促肾上腺皮质激素释放因子（CRF）诱导的p-tau/tau蛋白， 体外增加。tau的过度磷酸化导致毒性tau寡聚体，并最终形成 AD大脑中的神经元缠结（NFT）与认知能力下降密切相关（34）;因此， tau磷酸化是AD和tau蛋白病新治疗方法的关键靶点。报告基础上 sAPPα通过抑制激酶GSK 3 β的活性降低p-tau/t-tau比值（9），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 该项目旨在鉴定优化的小分子sAPPα增强，p-tau/t-tau降低化合物， 在小鼠AD模型中的认知，用于进一步开发作为AD的新疗法。另一个目标是 阐明sAPPα增强和相关的p- tau/t-tau比值。在目标1中，我们将测试F03和我们手头的类似物以及目标2中的新类似物。 SH-SY 5 Y细胞和3xTg-AD原代神经元，以确定sAPPα增强和p-tau/t-tau的EC 50 减少。将评价优先化合物的受体结合和APP结合。三级测试将 在来自AD受试者的诱导多能干细胞（iPSC）衍生的神经元中， 量化在目标2中，药物化学将用于设计新的化学实体（NCE）类似物， 与受体和APP结合的迭代方式沿着增强的药物样性质和口服脑 磁导率在目标3中，对类似物的ADME研究包括溶解性、微粒体稳定性、蛋白结合、平行 人工膜渗透性测定（PAMPA）分析和体内药代动力学（PK）。最优 候选人将接受安全性（包括hERG）和脱靶组分析沿着分析对 其他激酶和tau磷酸化位点。在目标4中，体内试验，包括对选择的急性研究 评价对脑室内（ICV）递送的CRF诱导的p-tau/t-tau增加的作用的候选物， 3xTg-AD小鼠的脑。活性候选药物将在3xTg-AD的慢性4周疗效研究中进行测试， ApoE 4 -5XFAD AD模型小鼠。读数将包括行为和组织病理学分析，包括 海马神经突负荷和神经元棘密度。生化结果包括p-tau/t-tau比值、sAPPα Aβ。磷酸蛋白质组学用于读数与认知之间相关性的机制研究。