Selective modulation of y-secretase processing through substrate binding

描述（由申请人提供）：项目负责人/主要研究者（最后一名、第一名、中间名）：Kukar，托马斯L.项目总结（见说明）：许多证据支持这一假设，即选择性靶向A42是预防和可能治疗老年痴呆症的主要原因阿尔茨海默病（AD）的理想治疗策略。一类化合物叫做？-分泌酶调节剂（GSM）正被用作AD治疗剂，因为它们能够选择性地改变A42水平。第一个被发现的GSM是某些非甾体抗炎药（NSAID），已显示其选择性降低A42而不全面抑制APR加工。作为整体，GSM最小地改变总Abeta产生，而不是转移γ-分泌酶切割位点。GSMs的机制尚未得到最终证实，对其活性提出了不同的解释，包括：1）变构结合？-分泌酶2）抑制Rho-ROCK信号传导途径3）早老素的构象变化或4）减少APR的二聚化。我们最近发现，使用新的GSM光亲和探针，这些药物不标记γ-分泌酶，而是通过结合底物来调节切割，我们假设，GSMs对APR的结合移动了APP-CTF在膜中的位置，导致γ-分泌酶切割的改变。这一假设将通过以下具体目标进行检验：1）使用分子生物学和蛋白质生物化学的组合，研究GSMs的底物靶向如何产生A？切割模式的转变2）确定GSMs通过？分泌酶与其他底物相比，使用质谱和3）测试非天然氨基酸是否可用于研究APP-CTF的蛋白水解？分泌酶目标1和职业/技术培训将在辅导阶段进行，这将有助于在独立阶段执行目标2和3。这些研究应该提供更多的洞察机制，NSAID和其他GSM移动A？裂解，以及它们如何发挥其体内保护作用。这项工作也将指导未来的努力，以设计更有效的GSM，这将是有用的化学探针，了解生物学？-分泌酶和作为阿尔茨海默病的潜在治疗剂。相关性（见说明书）：阿尔茨海默病（AD）是困扰老年人的最常见的痴呆症类型，目前尚无治愈方法。?-分泌酶调节剂（GSM）是正在研究的一类有希望的治疗AD的药物。拟议的研究将为GSM如何工作提供关键的见解，并可能最终导致改善AD治疗方法。

DESCRIPTION (provided by applicant): Program Director/Principal Investigator (Last, First, Middle): Kukar, Thomas L. PROJECT SUMMARY (See instructions): Numerous lines of evidence support the hypothesis that selective targeting of A¿42 is an ideal therapeutic strategy to prevent and possibly treat Alzheimer's disease (AD), the major cause of dementia among the elderly. A class of compounds called ?-secretase modulators (GSMs) is being pursued as AD therapeutics because they are able to selectively alter A¿42 levels>. The first GSMs to be discovered were certain non- steroidal anti-inflammatory drugs (NSAIDs), which have been shown to selectively decrease A¿42 without global inhibition of APR processing. As whole GSMs minimally alter total Abeta production and instead shift the gamma-secretase cleavage site. The mechanism of GSMs has not been conclusively proven and different explanations for their activity have been proposed including: 1) allosteric binding to ?-secretase 2) inhibition of the Rho-ROCK signaling pathway 3) conformational changes in presenilin or 4) decreased dimerization of APR. We have recently discovered, using novel GSM photoaffinity probes, that these drugs do not label the y-secretase enzyme but instead modulate cleavage by binding to a the substrate, APP. We hypothesize that binding of APR by GSMs shifts the} position of APP-CTF in the membrane resulting in altered gamma-secretase cleavage. This hypothesis will be tested through the following specific aims: 1) investigate how substrate targeting by GSMs produces a shift in the cleavage pattern of A¿ using a combination of molecular biology and protein biochemistry 2) determine the specificity of GSMs for affecting APP proteolysis by ?-secretase in comparison to other substrates using mass spectrometry and 3) test if unnatural amino acids can be used to study proteolysis of APP-CTF¿ by ?-secretase. Aim 1 and career/technical training will take place during the mentored phase, which will facilitate the execution of Aims 2 and 3 during the independent phase. These studies should provide additional insight into the mechanisms whereby NSAIDs and other GSMs shift A¿ cleavage and how they exert their protective effects in vivo. This work will also guide future efforts to design more potent GSMs which will be useful as chemical probes for understanding the biology of ?-secretase and as potential therapeutics for Alzheimer's disease. RELEVANCE (See instructions): Alzheimer's disease (AD) is the most common type of dementia afflicting the elderly with no known cure. ?- secretase modulators (GSMs) are a promising class of drugs under investigation to treat AD. The proposed research will provide critical insight into how GSMs work and may ultimately lead to improved AD therapeutics.