Development of Molecular Probe Inhibitors of Pathogenic, Cytosolic Cathespin B in Traumatic Brain Injury and Alzheimers Disease Neurodegeneration

创伤性脑损伤(TBI)和阿尔茨海默病(AD)会导致长期的行为缺陷和大脑 神经退行性变。目前还没有治疗脑外伤或阿尔茨海默病的有效药物；因此，在机制方面的进展。 基于对这些大脑疾病的了解，可以确定新的药物靶向方法。值得注意的是， 组织蛋白酶B被认为是参与行为功能障碍和神经病理的一种新机制 TbI和AD。组织蛋白酶B机制的前提是：(A)组织蛋白酶B在脑损伤和AD中升高 患者，(B)脑损伤和AD小鼠模型中组织蛋白酶B基因的敲除(KO)改善了行为缺陷 (C)组织蛋白酶B KO可减少脑神经病理。 颅脑损伤和AD导致溶酶体渗漏和组织蛋白酶B从溶酶体重新分布到胞浆 导致脑内细胞死亡和炎性IL-1β的激活。这些发现导致了这样的假设 胞浆组织蛋白酶B参与了脑外伤和AD的发病过程。为了检验这一假设，理想的做法是 抑制致病的胞浆组织蛋白酶，不影响其正常的溶酶体功能，具有pH选择性 作为分子探针的抑制剂。我们的数据显示，组织蛋白酶B显示出不同的多肽切割特性 中性胞浆pH与溶酶体酸性pH的比较。这些差异切割属性支持 胞浆与溶酶体组织蛋白酶B的选择性底物和多肽抑制剂的研究进展 该项目的目标将是开发中性胞液组织蛋白酶B的pH选择性抑制剂，比较 以酸性溶酶体组织蛋白酶B为分子探针评价其假想致病作用 在细胞溶酶体渗漏过程中胞浆组织蛋白酶B的表达，从而导致神经变性和 颅脑损伤和阿尔茨海默病的行为缺陷。目标1将评估组织蛋白酶B在中性条件下的选择性切割特性 和酸性PHS，由Global‘MSP-MS和Positive 使用合成组合文库(PSSCL)进行扫描，用于设计和测试pH选择多肽 底物。目标2将利用组织蛋白酶B的pH选择性分析来鉴定天然产物抑制剂，已实现 通过筛选海洋和陆地天然产物的收集，并评估选择性和效力。目标 3将开发组织蛋白酶B的多肽抑制剂，通过用以下物质修饰pH选择性多肽底物来实现 AOMK、CMK或VS组；将评估选择性和效力。多肽和天然产物抑制剂将 评估A-β和H_2O_2诱导的神经细胞溶酶体渗漏对细胞死亡和IL-1β水平的影响 和神经胶质细胞。目的4研究脑损伤和阿尔茨海默病模型小鼠体内组织蛋白酶B的溶酶体渗漏 关于时间进程、脑区、神经元和神经胶质细胞。组织蛋白酶B抑制剂，已知和最新 开发的抑制剂，将在溶酶体渗漏之前和期间给予，以评估 行为缺陷和神经病理学。新型抑制剂将有利地靶向致病性胞浆。 组织蛋白酶B，而不是正常的溶酶体组织蛋白酶B，用于脑损伤和AD治疗的未来发展。

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.