Determinants of Notch-Sparing Gamma-Secretase Inhibition

性状（由申请方提供）：4 kDa淀粉样蛋白？-蛋白质（A？）与阿尔茨海默病（AD）的发病机制密切相关。一个？是通过I型膜淀粉样蛋白的连续蛋白水解产生的？蛋白质前体（APP），首先在管腔/细胞外侧，由膜栓系？分泌酶，然后在跨膜结构域（TMD）内通过？分泌酶，一种膜包埋的蛋白酶复合物，含有多通道早老素作为催化组分。劈开？-分泌酶决定A？肽在C-末端，与较长形式的A？含有更多的TMD，并且更容易自组装成神经毒性聚集体。不过呢？分泌酶被认为是开发AD治疗剂的重要靶标，该酶切割多种其他I型整合膜蛋白，最显著的是Notch受体。Notch的蛋白水解伴随着其胞内结构域的释放， 一个信号通路的一部分，是许多类型的细胞分化事件的核心，并抑制这一途径与？-分泌酶抑制剂（GSI）会导致严重的毒性， 对于AD药物来说是不可接受的。然而，对APP选择性超过Notch的GSI（所谓的“Notch-sparing”GSI）的发现已经导致若干候选物进入临床试验。然而，这些化合物选择性地抑制蛋白酶复合物的方法是未知的，并且对它们的选择性机制的理解可能揭示用于开发更具选择性的试剂的策略。该项目的目标是确定底物和酶的决定因素，赋予选择性的APP维斯-？-维斯Notch of Notch-sparing吗分泌酶抑制剂。考虑到这一目标，提出了解决以下问题的具体目标：（1）APP和Notch的哪些区域/残基赋予Notch保留GSI的底物选择性？(2)Notch-sparing GSI的效力和选择性是什么？携带家族性AD突变的分泌酶？(3)保留缺口的GSI对PS1和PS2的效力和选择性是什么？分泌酶？这些化合物的APP/Notch选择性的底物和酶决定簇的鉴定应该提供与这些化合物结合的位置以及该结合如何导致赋予底物选择性的变构变化相关的重要信息。

DESCRIPTION (provided by applicant): The 4 kDa amyloid ?-protein (A?) is strongly implicated in the pathogenesis of Alzheimer's disease (AD). A? is produced through successive proteolysis of the type I integral membrane amyloid ?-protein precursor (APP), first on the lumen/extracellular side by the membrane-tethered ?-secretase and then within the transmembrane domain (TMD) by ?-secretase, a membrane-embedded aspartyl protease complex that contains the multi-pass presenilin as the catalytic component. Cleavage by ?-secretase determines the length of the A? peptide at the C-terminus, with longer forms of A? containing more of the TMD and being much more prone to self-assembly into neurotoxic aggregates. Although ?-secretase is considered an important target for the development of AD therapeutics, the enzyme cleaves a variety of other type I integral membrane proteins, most notably Notch receptors. Proteolysis of Notch with attendant release of its intracellular domain is part of a signaling pathway that is central to many types of cell differentiation events, and inhibition of this pathway with ?-secretase inhibitors (GSIs) results in severe toxicities that are unacceptable for an AD drug. Nevertheless, the discovery of GSIs that are selective for APP over Notch (so-called "Notch-sparing" GSIs) has led to the advancement of several candidates into clinical trials. However, the means by which these compounds selectively inhibit the protease complex are unknown, and an understanding of their mechanisms of selectivity might reveal strategies for developing more selective agents. The goal of this project is to identify substrate and enzyme determinants that confer selectivity for APP vis-?-vis Notch of Notch-sparing ?-secretase inhibitors. With this goal in mind, specific aims are proposed to address the following questions: (1) What regions/residues of APP and Notch confer substrate selectivity of Notch-sparing GSIs? (2) What is the potency and selectivity of Notch-sparing GSIs for ?-secretase carrying familial AD mutations? (3) What is the potency and selectivity of Notch-sparing GSIs for PS1 vs. PS2 ?-secretase? The identification of substrate and enzyme determinants of APP/Notch selectivity for these compounds should provide important information relevant to where these compounds bind and how that binding might result in allosteric changes that confer substrate selectivity.