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 我们先前在固体淀粉样蛋白前体蛋白α（SAPPα）增强作用上报道了（2） Tropisetron（F03）。高度可渗透的F03在49个国家 /地区批准用于治疗术后 恶心和呕吐（PONV），是一种多功能配体：它是潜在的5-HT3 5-羟色胺受体（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 到淀粉样前体蛋白（EAPP）的细胞外结构域。在我们的研究中，我们发现F03增加了SAPPα 并显着降低了两个阿尔茨海默氏病（AD）中的磷-TAU（P-TAU）/TAU（T-TAU）比率 鼠标模型。我们表明，F03还可以减少皮质激素释放因子（CRF）诱导的p-tau/tau 体外增加。 tau的高磷酸化导致有毒的tau低聚物，并最终形成 AD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Tauopath的新治疗方法的关键靶标。基于报告 通过抑制激酶GSK3β的活性，SAPPα降低了P-TAU/T-TAU的比率（9），我们计划 评估我们的SAPPα增强子在体外和体内降低P-TAU/T-TAU比率的能力。一个关键目标 项目旨在鉴定优化的小分子SAPPα增强，p-tau/t-tau降低化合物，以改善 鼠类广告模型中的认知，作为一种新的AD疗法，以进一步发展。另一个目标是 阐明SAPPα增强的基本作用机理（MOA）和P-的相关降低 tau/t-tau比率。在AIM 1中，我们将测试F03和类似物，以及来自AIM 2的新类似物 SH-SY5Y细胞和3XTG-AD原发性神经元，以建立用于SAPPα增强和p-tau/t-tau的EC50 减少。将评估优先化的化合物的受体结合和应用结合。三级测试将 在诱导的多能干细胞（IPSC）衍生的AD受试者中，包括突触脊柱密度 定量。在AIM 2中，医学化学将用于设计新的化学实体（NCE）类似物 具有受体和应用结合的迭代方式，以及增强的药物样特性和口服大脑 渗透性。在AIM 3中，对类似物的ADME研究包括溶解度，微粒体稳定性，蛋白质结合，平行 人造膜通透性评估（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）和脱离目标面板分析，并分析对 其他激酶和tau磷酸化位点。在AIM 4体内测试中，包括针对选择的急性研究 评估对脑室内（ICV）递送的CRF诱导P-TAU/T-TAU的影响的候选者增加 3XTG-AD小鼠中的大脑。活跃候选者将在3XTG-AD的慢性4周效率研究中进行测试， APOE4-5XFAD AD模型小鼠。读数将包括行为和组织病理学分析，包括 海马神经蛋白负荷和神经脊柱密度。生化结果包括p-tau/t-tau比，sappα 和Aβ。通过磷蛋白质组学进行的机械研究，用于读数与认知之间的相关性。