Gamma Secretase Assays to Discover Drugs for Alzheimer's

描述（由申请人提供）：阿尔茨海默病（AD）是一种导致记忆和认知功能丧失的长期衰弱性疾病。目前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中在细胞外淀粉样斑块中积累的分泌的Abeta肽的主要来源。 然而，以前的γ-分泌酶抑制剂筛选仅分析了Abeta肽产生的次要组成性分泌途径。与组成性分泌途径相比，不同的蛋白酶存在于调节性分泌途径中。显然，调节分泌途径中的γ-分泌酶必须被靶向用于药物抑制以提供最大程度的Abeta降低。因此，I期项目开发了用于产生Abeta的调节分泌囊泡中的γ-分泌酶的高通量测定。确定了γ-分泌酶的候选抑制剂，这将为该II期项目的“聚焦”和“优化”文库的设计和合成提供基础。I期结果还表明找到与γ-分泌酶-40相比选择性抑制γ-分泌酶-42的药剂的可行性。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测定法，用于测定受调节的分泌途径中的Abeta。该II期项目的目标将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。此类抑制剂将被视为先导化合物。在具体目标1中，将在高通量体外试验中筛选“集中”文库化合物的努力，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，将基于来自“聚焦”文库的抑制剂的结构特征设计和合成进一步的“优化”文库，在高通量测定中筛选，并测试在神经元嗜铬细胞的调节分泌途径中Abeta的减少。在第三个目标中，先导化合物将在脑神经元细胞中进行评估，以鉴定在受调节的分泌途径中减少Abeta肽产生的化合物。结果可能会确定新的先导化合物，为未来的临床前动物研究开发有效的药物治疗AD。

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.