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.

项目概要/摘要 我们之前报道过 (2) 可溶性淀粉样前体蛋白 α (sAPPα) 的增强作用 托烷司琼(F03)。高脑渗透性 F03 已在 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-HT3 血清素受体 (5-HT3R) 拮抗剂 (Ki ~ 3 nM)、部分 α7 烟碱乙酰胆碱受体 (α7nAChR) 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 还显着降低两种阿尔茨海默病 (AD) 中的磷酸 tau (p-tau)/总 tau (t-tau) 比率 鼠标模型。我们发现 F03 还可以减少促肾上腺皮质激素释放因子 (CRF) 诱导的 p-tau/tau 体外增加。 tau 蛋白的过度磷酸化会导致有毒的 tau 寡聚体并最终形成 AD 大脑中的神经原纤维缠结 (NFT) 与认知能力下降密切相关 (34)；因此，减少 tau 磷酸化是 AD 和 tau 病新治疗方法的关键靶点。根据报告 sAPPα 通过抑制激酶 GSK3β 的活性来降低 p-tau/t-tau 比率 (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。该计划的一个关键目标是 该项目旨在确定优化的小分子 sAPPα 增强、p-tau/t-tau 降低化合物，以改善 小鼠 AD 模型中的认知，进一步开发为 AD 的新疗法。另一个目标是 阐明 sAPPα 增强和 p-相关减少的潜在作用机制 (MOA) tau/t-tau 比率。在目标 1 中，我们将测试 F03 和我们现有的类似物以及目标 2 中的新类似物 SH-SY5Y 细胞和 3xTg-AD 原代神经元建立 sAPPα 增强和 p-tau/t-tau 的 EC50 减少。 Prioritized compounds would be evaluated for receptor binding and APP binding.第三次测试将 存在于 AD 受试者的诱导多能干细胞 (iPSC) 衍生的神经元中，包括突触棘密度 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 受体和 APP 结合的迭代方式以及增强的药物样特性和口腔大脑 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 人工膜通透性测定（PAMPA）分析和体内药代动力学（PK）。最佳的 候选人将接受安全性（包括 hERG）和脱靶小组分析以及对 其他激酶和 tau 磷酸化位点。在 Aim 4 体内测试中，包括针对特定对象的急性研究 候选者评估对脑室内 (ICV) 递送的 CRF 诱导的 p-tau/t-tau 增加的影响 3xTg-AD 小鼠的大脑。积极的候选者将在 3xTg-AD 和 3xTg-AD 中接受为期 4 周的长期疗效研究的测试 ApoE4-5XFAD 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 比率、sAPPα 和 Aβ。磷酸蛋白质组学的机制研究用于读数和认知之间的相关性。