Gamma Secretase Assays to Discover Drugs for Alzheimer's

描述(申请人提供)：阿尔茨海默病(AD)是一种长期的、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药物来改善AD的症状。阿尔茨海默病的重要分子药物靶点是在AD中产生神经毒性Aβ多肽的伽玛分泌酶。阿尔茨海默病患者细胞外淀粉样蛋白斑块中积聚的Abeta多肽主要来源于神经元调节的分泌途径。 然而，以前对伽马分泌酶抑制剂的筛选只分析了产生Abeta肽的次要的结构性分泌途径。与结构性分泌途径相比，调节分泌途径中存在不同的蛋白酶。显然，调节分泌途径中的伽马分泌酶必须作为药物抑制的靶点，以提供最大程度的Abeta减少。因此，第一阶段项目开发了高通量检测产生Abeta的调节分泌囊泡中的伽马分泌酶。确定了一种候选的伽马分泌酶抑制剂，这将为设计和合成这一第二阶段项目的“重点”和“优化”文库提供基础。第一阶段的结果还表明，与伽马分泌酶-40相比，寻找选择性抑制伽马分泌酶-42的药物是可行的。第一阶段的项目还开发了神经元嗜铬细胞和大脑皮层神经元对调节分泌途径中的Abeta的分析。这个第二阶段项目的目标是利用调节的分泌囊泡作为神经细胞中产生Aβ多肽的主要部位和靶点，以识别与伽马分泌酶-40相比选择性抑制伽马分泌酶-42的抑制分子。此类抑制剂将被视为先导化合物。在具体目标1中，将在高通量的体外试验中筛选“集中”的库化合物，以确定嗜铬小泡中的伽马分泌酶-40和伽马分泌酶-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”库中抑制剂的结构特征设计和合成进一步的“优化”文库，在高通量分析中进行筛选，并测试神经元嗜铬细胞调节分泌途径中Abeta的还原情况。在第三个目标中，先导化合物将在脑神经细胞中进行评估，以确定在调节的分泌途径中减少Abeta多肽产生的化合物。结果可能会为未来的临床前动物研究确定新的先导化合物，以开发治疗AD的有效药物。

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.