Gamma Secretase Assays to Discover Drugs for Alzheimer's

伽马分泌酶检测发现治疗阿尔茨海默病的药物

• 批准号: 6989920
• 负责人: GREGORY R HOOK
• 金额: $ 76.33万
• 依托单位: AMERICAN LIFE SCIENCE PHARMACEUTICALS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6989920
• 关键词: Alzheimer' s disease; amyloid proteins; animal tissue; aspartic endopeptidases; bioassay; blood brain barrier; chemical structure function; chromaffin cells; drug discovery /isolation; drug screening /evaluation; enzyme activity; fluorescent dye /probe; high throughput technology; neuritic plaques; neurons; neuropharmacology; nicotine; potassium chloride; protease inhibitor; technology /technique development; tissue /cell culture; vesicle /vacuole

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a long-term, debilitating disorder that causes loss of memory and cognitive functions. There is an urgent need for effective therapeutic agents to ameliorate the symptoms of AD. Important molecular drug targets in AD are the gamma-secretases that produce the neurotoxic Abeta peptides in AD. The regulated secretory pathway of neurons represents the major source of secreted Abeta peptides that accumulate in extracellular amyloid plaques in AD. However, previous screens for inhibitors of gamma-secretase have only analyzed the minor constitutive secretory pathway for Abeta peptide production. Different proteases are present in the regulated secretory pathway compared to the constitutive secretory pathway. Clearly, gamma-secretase in the regulated secretory pathway must be targeted for drug inhibition to provide the greatest reduction of Abeta. Therefore, the Phase I project developed high-throughput assays for gamma-secretases in regulated secretory vesicles that produce Abeta. A candidate inhibitor of gamma-secretases was identified, which will provide the basis for design and synthesis of 'focused' and 'optimized' libraries for this Phase II project. Phase I results also indicate feasibility to find agents that selectively inhibit gamma-secretases-42 compared to gamma-secretases-40. The Phase I project also developed neuronal chromaffin cell and brain cortical neuron assays for Abeta in the regulated secretory pathway. The goal of this phase II project will be to utilize the regulated secretory vesicle as the major site and target of Abeta peptide production in neuronal cells, for identifying inhibitor molecules that selectively inhibit gamma-secretases-42 compared to gamma-secretases-40. Such inhibitors will be considered as lead compounds. In specific aim 1, efforts for 'focused' library compounds will be screened in the high throughput in vitro assays to identify inhibitors of gamma-secretases-40 and gamma-secretases-42 in chromaffin vesicles; inhibitors will then be tested in chromaffin cells for reduction of Abeta. In the second aim, further 'optimized' libraries will be designed and synthesized based on structural features of inhibitors from the 'focused' library, screened in the high throughput assays, and tested for reduction of Abeta in the regulated secretory pathway of neuronal chromaffin cells. In the third aim, lead compounds will undergo evaluation in brain neuronal cells to identify compounds that reduce production of Abeta peptides in the regulated secretory pathway. Results will likely identify novel lead compounds for future pre-clinical animal studies for development of effective drugs for AD.

描述（由申请人提供）：阿尔茨海默病（AD）是一种长期的、使人衰弱的疾病，会导致记忆和认知功能丧失。迫切需要有效的治疗剂来改善AD的症状。 AD 中重要的分子药物靶标是在 AD 中产生神经毒性 Abeta 肽的 γ 分泌酶。神经元的调节分泌途径代表了 AD 细胞外淀粉样斑块中积累的分泌 Abeta 肽的主要来源。 然而，先前对γ-分泌酶抑制剂的筛选仅分析了Abeta肽产生的次要组成型分泌途径。与组成型分泌途径相比，调节性分泌途径中存在不同的蛋白酶。显然，药物抑制必须针对受调节的分泌途径中的γ-分泌酶，以最大程度地减少Abeta。因此，第一阶段项目开发了对产生 Abeta 的调节分泌囊泡中的 γ 分泌酶的高通量检测。确定了一种候选的γ-分泌酶抑制剂，这将为该二期项目的“重点”和“优化”库的设计和合成提供基础。 I 期结果还表明寻找与 γ-分泌酶-40 相比选择性抑制 γ-分泌酶-42 的药物的可行性。第一阶段项目还开发了神经元嗜铬细胞和大脑皮层神经元检测，以检测调节分泌途径中的 Abeta。该 II 期项目的目标是利用受调节的分泌囊泡作为神经元细胞中 Abeta 肽产生的主要位点和靶点，以鉴定与 gamma-secretases-40 相比选择性抑制 gamma-secretases-42 的抑制剂分子。此类抑制剂将被视为先导化合物。在具体目标 1 中，将在高通量体外测定中筛选“重点”库化合物，以鉴定嗜铬囊泡中 γ-分泌酶 40 和 γ-分泌酶 42 的抑制剂；然后将在嗜铬细胞中测试抑制剂以减少 Abeta。在第二个目标中，将根据“聚焦”文库中抑制剂的结构特征设计和合成进一步“优化”的文库，在高通量测定中进行筛选，并测试神经元嗜铬细胞调节分泌途径中 Abeta 的减少。第三个目标是，先导化合物将在脑神经元细胞中进行评估，以确定能够减少受调节分泌途径中 Abeta 肽产生的化合物。结果可能会确定新的先导化合物，用于未来临床前动物研究，以开发治疗 AD 的有效药物。