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: 7101003
• 负责人: GREGORY R HOOK
• 金额: $ 80.84万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7101003
• 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）：阿尔茨海默氏病（AD）是一种长期，令人衰弱的疾病，会导致记忆和认知功能的丧失。迫切需要有效的治疗剂来改善AD的症状。 AD中的重要分子药物靶标是在AD中产生神经毒性Abeta肽的γ-分泌酶。神经元的受调节分泌途径代表了在AD中积累的分泌Abeta肽的主要来源。 但是，以前的伽马分泌酶抑制剂的筛选仅分析了用于ABETA肽产生的次要构成分泌途径。与本构分泌途径相比，调节的分泌途径中存在不同的蛋白酶。显然，必须针对药物抑制作用的伽马分泌酶以提供ABETA的最大降低。因此，I期项目在产生Abeta的受调节分泌囊泡中开发了对伽马分泌酶的高通量测定法。确定了γ-分泌酶的候选抑制剂，这将为该II期项目的“集中”和“优化”库的设计和综合提供基础。第一阶段的结果还表明，与γ-分泌酶-40相比，可以选择性地抑制γ-分泌酶-42的药物可行性。 I期项目还开发了在受调节分泌途径中的Abeta的神经元铬蛋白细胞和脑皮质神经元测定。该第二阶段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标，用于鉴定与γ-分泌酶-40相比，选择性抑制γ-分泌酶42的抑制剂分子。这种抑制剂将被视为铅化合物。在特定的目标1中，将在高吞吐量的体外测定中筛选“聚焦”库化合物的努力，以鉴定铬脂囊泡中γ-分泌酶-40和γ-分泌酶-42的抑制剂。然后，将在铬蛋白细胞中测试抑制剂，以减少ABETA。在第二个目标中，将根据“聚焦”库的抑制剂的结构特征设计和合成进一步的“优化”文库，并在高吞吐量测定中进行筛选，并在神经元铬素细胞的受调节分泌途径中进行了降低ABETA的测试。在第三个目标中，铅化合物将在脑神经元细胞中进行评估，以鉴定降低受调节分泌途径中Abeta肽产生的化合物。结果可能会确定未来临床前动物研究的新型铅化合物，以开发有效的AD药物。