Molecular Probes for Alzheimer's Gamma-Secretase

描述（由申请人提供）：淀粉样蛋白-3肽（A？）生产的最后一步，阿尔茨海默病（AD）特征性脑斑块的主要成分是A-3前体蛋白（APR）的膜内蛋白水解，分泌酶这种蛋白酶由于其复杂性而长期难以鉴定。尽管如此，在过去的几年里，我们已经看到了相当大的进展，了解生物化学？分泌酶和小分子抑制剂已成为不可或缺的工具。具体来说，我们和其他人发现（1）？分泌酶可被底物类似物抑制，这些底物类似物含有乙酰蛋白酶过渡态模拟物;（2）在家族性AD中突变的多通道早老素（PS）含有两个保守的催化必需的跨膜受体;（3）PS是？-分泌酶;（4）亲和纯化？-分泌酶活性与固定化抑制剂分离不仅PS，但也有其他三个必要的活性成分：Nicastrin（NCT），Aph- 1，和Pen-2;（5）蛋白酶显然具有一个初始对接点的外部部分的基本信号传导途径在细胞分化和胚胎发生的基板。?-分泌酶是一个新兴的膜内蛋白酶家族的创始成员，其活性位点显然嵌入脂质双层中。尽管取得了显着的进展，仍然有很多关于这种不寻常的和重要的蛋白酶未知。为了推进对机械的理解-分泌酶和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？分泌酶活性部位？基板对接点的位置？(2)怎么样？-分泌酶能识别并处理底物吗？(3)怎么可能？-分泌酶被别构调节以阻断APP而不是Notch的蛋白水解？总结：这个项目的目标是了解？分泌酶，一种对阿尔茨海默病的病因至关重要的复合酶。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，并探索其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.