Mechanism of gamma-secretase modulators

摘要 阿尔茨海默病（AD）是一种破坏性的神经退行性疾病，目前影响5.8 数百万美国人然而，目前没有FDA批准的治疗方法能够延迟或停止 疾病进展。β淀粉样蛋白（Aβ）斑块被认为是AD发病机制的组成部分，通过其自身的免疫反应。 在“淀粉样蛋白级联假说”中的作用，其中Aβ肽的积累启动了一条链， 导致神经变性和最终AD的病理事件。γ-分泌酶是一种酰基蛋白酶 负责处理各种基板。它被认为是一个有吸引力的药物靶点，因为它 在蛋白水解的最后一步中切割淀粉样前体蛋白（APP），产生不同 此外，APP和早老素（γ-分泌酶的催化亚基）的突变与 家族性AD并显示改变Aβ产生。不幸的是，所有γ-分泌酶抑制剂的临床试验都证明， 由于对其他γ-分泌酶底物（如Notch）的脱靶效应而不成功。γ- 分泌酶抑制剂刺激了一系列新化合物的发现和开发，称为γ- 分泌酶调节剂（GSM）。最初发现NSAID的一个子集可以选择性地降低 致病性Aβ42有利于致病性较低的Aβ38，而不抑制Notch。这些方面的改进 第二代GSM的诞生。该项目将重点关注两类第二代 GSMs：源自NSAID和咪唑GSMs的酸性GSMs。酸和咪唑GSM已经被 以多组为特征，并显示出减少致病性Aβ种类，同时增加 致病性较低的物种。然而，他们在关于Aβ的临床试验中仍然表现出局限性， 选择性、效力和毒性。我们的目标是确定GSM的分子机制， 改进它们用于AD药物开发。利用基于GSM的光探针标记γ-分泌酶， 发现酸性和咪唑类GSMs与γ-分泌酶早老素亚基上的不同位点结合。基于 基于这一信息，我们假设酸和咪唑GSMs与γ- 分泌酶和协同作用，有效地调节Aβ的产生。使用接口的方法， 本项目的主要目的是：1）确定GSMs与γ-分泌酶结合的机制 和2）确定GSM组合对Aβ产生、毒性和认知的影响。探讨 GSMs的分子机制，我们将使用化学探针结合质谱和结构 建模以识别精确的结合位点。我们还将评估Aβ- 分泌细胞系，并使用最有效的浓度来分析毒性和认知性能， AD小鼠模型。总的来说，拟议的研究将使我们能够解码GSM的机制， 最终，开发出具有最高选择性和效力的GSM。这项提案的结果将开启新的 调节γ-分泌酶以更有效地治疗AD的可能性。

Abstract Alzheimer’s disease (AD) is a devastating neurodegenerative disorder which currently affects 5.8 million Americans. However, there are currently no FDA-approved treatments capable of delaying or stopping disease progression. Amyloid-beta (Aβ) plaques are believed to be integral to AD pathogenesis through their role in the “amyloid cascade hypothesis,” in which the accumulation of Aβ peptides initiates a chain of pathological events leading to neurodegeneration and ultimately AD. γ-secretase is an aspartyl protease responsible for processing a wide range of substrates. It is considered an attractive drug target because it cleaves amyloid precursor protein (APP) in the final step of proteolysis to generate Aβ peptides of varying lengths; moreover, mutations in APP and presenilin, the catalytic subunit of γ-secretase, have been linked to familial AD and shown to alter Aβ production. Unfortunately, all clinical trials of γ-secretase inhibitors proved unsuccessful due to off target effects on other γ-secretase substrates, such as Notch. The failures of γ- secretase inhibitors spurred the discovery and development of a new series of compounds known as γ- secretase modulators (GSMs). A subset of NSAIDs were initially discovered to selectively reduce levels of the pathogenic Aβ42 in favor of the less pathogenic Aβ38 without inhibition of Notch. Improvements on these compounds led to second generation GSMs. This project will focus on two classes of second generation GSMs: acid GSMs derived from NSAIDs and imidazole GSMs. Acid and imidazole GSMs have been characterized by multiple groups and showed promise in decreasing pathogenic Aβ species while increasing the less pathogenic species. However, they still demonstrated limitations in clinical trials regarding Aβ selectivity, potency, and toxicity. Our objective is to define the molecular mechanism of GSMs in order to improve them for AD drug development. Using GSM-based photoprobes to label γ-secretase, our laboratory has found that acid and imidazole GSMs bind to distinct sites on the presenilin subunit of γ-secretase. Based on this information, we hypothesize that acid and imidazole GSMs bind to distinct, allosteric sites of γ- secretase and work in synergy to effectively modulate Aβ production. Using approaches at the interface of biology and chemistry, this project aims to: 1) Determine the mechanism of binding of GSMs on γ-secretase and 2) Determine the effects of GSM combination on Aβ production, toxicity, and cognition. To investigate the molecular mechanisms of GSMs, we will use chemical probes combined with mass spectrometry and structural modeling to identify the precise binding sites. We will also evaluate pairs of acid and imidazole GSMs in Aβ- secreting cell lines and use the most effective concentrations to analyze toxicity and cognitive performance in an AD mouse model. Overall, the proposed studies will enable us to decode the mechanism of GSMs and, ultimately, develop GSMs with the highest selectivity and potency. Findings from this proposal will unlock new possibilities for modulation of γ-secretase to more effectively treat AD.