Development of molecular probe inhibitors of pathogenic, cytosolic cathespin B in traumatic brain injury and Alzheimers Disease neurodegeneration

开发创伤性脑损伤和阿尔茨海默病神经变性中致病性胞质组织蛋白酶 B 的分子探针抑制剂

• 批准号: 10199079
• 负责人: Vivian Y. H Hook
• 金额: $ 74.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10199079
• 关键词: Affect; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease therapeutic; Amyloid beta-42; Amyloid beta-Protein; Apolipoprotein E; Behavioral; Biological Assay; Brain; Brain Diseases; Brain Injuries; Brain region; Cathepsins; Cathepsins B; Cell Death; Chemicals; Cleaved cell; Collection; Cysteine; Cytosol; Data; Development; Enzymes; Evaluation; Extravasation; Functional disorder; Future; Genes; Goals; Hydrogen Peroxide; Immunofluorescence Immunologic; Inflammatory; Interleukin-1 beta; Ketones; Knock-out; Lead; Libraries; Location; Lysosomes; Mass Spectrum Analysis; Memory impairment; Modeling; Modification; Molecular Probes; Monitor; Natural Products; Nerve Degeneration; Neuroglia; Neurons; Oxidative Stress; Pathogenesis; Pathogenicity; Patients; Peptide Hydrolases; Peptides; Play; Property; Protease Inhibitor; Role; Scanning; Site; Structure; Testing; Therapeutic Agents; Time; Traumatic Brain Injury; abeta accumulation; base; brain cell; cell injury; combinatorial; controlled cortical impact; design; divinyl sulfone; drug development; fluorescence imaging; improved; in vivo; inhibitor/antagonist; innovation; knockout gene; motor disorder; mouse model; neuropathology; new therapeutic target; novel; novel therapeutic intervention; protein aminoacid sequence; screening; tau Proteins; therapeutically effective; tool

Traumatic brain injury (TBI) and Alzheimer's disease (AD) result in long-term behavioral deficits and brain neurodegeneration. There are no effective therapeutic agents for TBI or AD; therefore, advances in mechanism- based understanding of these brain disorders can identify new drug targeting approaches. Significantly, cathepsin B has been shown as a novel mechanism participating in behavioral dysfunctions and neuropathology of TBI and AD. The premise for the cathepsin B mechanism is that (a) cathepsin B is elevated in TBI and AD patients, (b) knockout (KO) of the cathepsin B gene in TBI and AD mouse models improves deficits in behavioral dysfunctions, respectively, and (c) cathepsin B KO reduces brain neuropathology. TBI and AD results in lysosomal leakage and redistribution of cathepsin B from lysosomes to the cytosol to result in cell death and activation of inflammatory IL-1β in brain. These findings lead to the hypothesis that cytosolic cathepsin B participates in the pathogenesis of TBI and AD. To test this hypothesis, it will be ideal to inhibit the pathogenic cytosolic cathepsin, without affecting its normal lysosomal function, with pH selective inhibitors as molecular probes. Our data shows that cathepsin B displays different peptide cleavage properties at neutral cytosolic pH compared to lysosomal acidic pH. These differential cleavage properties support the development of selective substrates and peptide inhibitors of cytosolic compared to lysosomal cathepsin B. The goal of this project will be to develop pH selective inhibitors of neutral cytosolic cathepsin B, compared to acidic lysosomal cathepsin B, as molecular probes for evaluation of the hypothesized pathogenic role of cytosolic cathepsin B during cellular lysosomal leakage which leads to neurodegeneration and behavioral deficits of TBI and AD. Aim 1 will assess the selective cleavage properties of cathepsin B at neutral and acidic pHs, achieved by global 'Multiplex Substrate Profiling Mass Spectrometry (MSP-MS) and positional scanning using a synthetic combinatorial library (PSSCL), for design and testing of pH selective peptide substrates. Aim 2 will utilize pH selective assays of cathepsin B to identify natural product inhibitors, achieved by screening collections of marine and terrestrial natural products, and assessing selectivity and potency. Aim 3 will develop peptidic inhibitors of cathepsin B, achieved by modifying pH selective peptide substrates with AOMK, CMK, or VS groups; selectivity and potency will be assessed. Peptidic and natural product inhibitors will be assessed for effects on cell death and IL-1β levels during Aβ- and H2O2-induced lysosomal leakage in neurons and glial cells. Aim 4 will characterize in vivo lysosomal leakage of cathepsin B in TBI and AD mouse models with respect to time-course, brain regions, and neuronal and glial cells. Cathepsin B inhibitors, known and newly developed inhibitors, will be given before and during lysosomal leakage for evaluation of improvements in behavioral deficits and neuropathology. The novel inhibitors will advantageously target pathogenic cytosolic cathepsin B, rather than normal lysosomal cathepsin B, for future development of TBI and AD therapeutics.

创伤性脑损伤 (TBI) 和阿尔茨海默病 (AD) 会导致长期行为缺陷和大脑损伤 神经退行性疾病尚无有效的治疗药物，因此，机制方面取得了进展。 重要的是，对这些大脑疾病的了解可以确定新的药物靶向方法。 组织蛋白酶 B 已被证明是一种参与行为功能障碍和神经病理学的新机制 组织蛋白酶 B 机制的前提是 (a) 组织蛋白酶 B 在 TBI 和 AD 中升高。 (b) TBI 和 AD 小鼠模型中组织蛋白酶 B 基因敲除 (KO) 可改善行为缺陷 (c) 组织蛋白酶 B KO 分别降低脑神经病理学。 TBI 和 AD 导致溶酶体渗漏和组织蛋白酶 B 从溶酶体重新分布到细胞质中 导致细胞死亡和大脑中炎症性 IL-1β 的激活。这些发现导致了这样的假设： 胞质组织蛋白酶 B 参与 TBI 和 AD 的发病机制。为了验证这一假设，最好进行以下测试。 抑制致病性胞质组织蛋白酶，不影响其正常溶酶体功能，具有 pH 选择性 我们的数据显示组织蛋白酶 B 显示不同的肽切割特性。 与溶酶体酸性 pH 值相比，这些不同的裂解特性支持了中性胞质 pH 值。 与溶酶体组织蛋白酶 B 相比，开发胞质的选择性底物和肽抑制剂。 该项目的目标是开发中性胞质组织蛋白酶 B 的 pH 选择性抑制剂 酸性溶酶体组织蛋白酶 B，作为评估其致病作用的分子探针 细胞溶酶体渗漏过程中胞质组织蛋白酶 B 的产生，导致神经变性和 TBI 和 AD 的行为缺陷目标 1 将评估组织蛋白酶 B 在中性时的选择性裂解特性。 和酸性 pH 值，通过全球“多重底物分析质谱 (MSP-MS)”和定位技术实现 使用合成组合库 (PSSCL) 进行扫描，用于设计和测试 pH 选择性肽 目标 2 将利用组织蛋白酶 B 的 pH 选择性测定来鉴定天然产物抑制剂。 通过筛选海洋和陆地天然产物的集合，并评估选择性和效力。 3将开发组织蛋白酶B的肽抑制剂，通过用以下物质改变pH选择性肽底物来实现 将评估 AOMK、CMK 或 VS 组；肽和天然产物抑制剂的选择性和效力。 评估 Aβ 和 H2O2 诱导的神经元溶酶体渗漏期间对细胞死亡和 IL-1β 水平的影响 目标 4 将表征 TBI 和 AD 小鼠模型中组织蛋白酶 B 的体内溶酶体渗漏。 关于时间进程、大脑、神经元和神经胶质细胞的已知和新抑制剂。 开发的抑制剂，将在溶酶体渗漏之前和期间给予，以评估溶酶体渗漏的改善情况 新型抑制剂将有利地针对致病性胞质。 组织蛋白酶 B，而不是正常的溶酶体组织蛋白酶 B，用于未来 TBI 和 AD 疗法的开发。