Molecular Probes for Alzheimer's Gamma-Secretase

描述（由申请人提供）：淀粉样蛋白-3肽（A?）是阿尔茨海默病（AD）特征性脑斑块的主要成分，其生产的最后一步是-分泌酶对A-3前体蛋白（APR）的膜内蛋白水解。由于其复杂性，这种蛋白酶长期无法被识别。尽管如此，在过去的几年里，我们在了解生物化学方面取得了相当大的进展。-分泌酶和小分子抑制剂是实现这一目标不可或缺的工具。具体来说，我们和其他人已经发现(1)？-分泌酶被含有天冬氨酸蛋白酶过渡态模拟物的底物类似物抑制；(2)家族性AD中突变的多通道早老素（multi-pass Presenilins， PS）含有两个保守的跨膜天冬氨酸，对催化作用至关重要；(3) PS是-分泌酶过渡态类似物抑制剂的靶标；(4)亲和纯化？-分泌酶活性与固定化抑制剂分离不仅PS，但其他三个必需的活性成分：Nicastrin (NCT), Aph- 1，和Pen-2；(5)该蛋白酶在细胞分化和胚胎发生的重要信号通路的外侧，显然具有与底物的初始对接位点。？-Secretase是一个新兴的膜内蛋白酶家族的创始成员，其活性位点明显嵌入脂质双分子层。尽管取得了显著的进展，但对这种不寻常且重要的蛋白酶仍有许多未知之处。推进对？-分泌酶并探索其作为治疗靶点的潜力，我们提出了新的底物和抑制剂作为分子探针。这些新的分子探针旨在解决三个特定的目标：(1)什么是性质的？-分泌酶活性部位？底物对接的位置？(2)如何？分泌酶识别和处理底物？(3)怎么能？-分泌酶被变构调节以阻断APP的蛋白水解，而Notch？概要：这个项目的目标是理解？-分泌酶，这是一种复杂的酶，对阿尔茨海默病的病因至关重要。小的有机分子将被用作探针来表征这种酶，阐明它是如何工作的，并探索它作为治疗靶点的潜力。

DESCRIPTION (provided by applicant): The last step in the production of the amyloid--3 peptide (A?), the primary component of the characteristic cerebral plaques of Alzheimer's disease (AD), is intramembranous proteolysis of the A-3 precursor protein (APR) by ?-secretase. This protease long eluded identification because of its complexity. Nevertheless, the past few years have seen considerable progress toward understanding the biochemistry of ?-secretase, and small molecule inhibitors have been indispensable tools to this end. Specifically, we and others have found that (1) ?-secretase is inhibited by substrate-based analogues containing aspartyl protease transition-state mimics; (2) the multi-pass Presenilins (PS), mutated in familial AD, contain two conserved transmembrane aspartates essential for catalysis; (3) PS is the target of transition-state analogue inhibitors of ?-secretase; (4) affinity purification of ?-secretase activity with an immobilized inhibitor isolates not only PS, but also three other components essential for activity: Nicastrin (NCT), Aph- 1, and Pen-2; (5) the protease apparently possesses an initial docking site for substrate on the outer part of an essential signaling pathway in cell differentiation and embryogenesis. ?-Secretase is a founding member of an emerging family of intramembrane proteases that apparently have their active sites embedded in the lipid bilayer. Despite the remarkable progress, much remains unknown about this unusual and important protease. Toward advancing the mechanistic understanding of ?-secretase and exploring its potential as a therapeutic target, we propose new substrates and inhibitors as molecular probes. These new molecular probes are designed to address three specific aims: (1) What is the nature of the ?- secretase active site? Of the substrate docking site? (2) How does ?-secretase recognize and handle its substrates? (3) How can ?-secretase be allosterically modulated to block proteolysis of APP but not Notch? Lay Summary: The goal of this project is to understand ?-secretase, a complex enzyme critical to the cause of Alzheimer's disease. Small organic molecules will be used as probes to characterize this enzyme, elucidate how it works, and explore its potential as a therapeutic target.