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，其产生的最后一步是通过？-分泌酶对A-3前体蛋白(APR)进行膜内蛋白分解。由于其复杂性，这种蛋白水解酶长期以来一直未能被识别。然而，在过去的几年里，人们在了解β-分泌酶的生物化学方面取得了相当大的进展，小分子抑制剂已经成为实现这一目标不可或缺的工具。具体地说，我们和其他人发现：(1)β-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)在家族性AD中突变的多代早老素(PS)含有两个对催化至关重要的保守的跨膜天冬氨酸；(3)PS是β-分泌酶的过渡态类似物抑制剂的靶标；(4)用固定化的抑制剂亲和纯化β-分泌酶的活性不仅包括PS，而且还有其他三种活性必需的成分：尼古丁(NCT)、APH-1和PEN-2；(5)在细胞分化和胚胎发育的重要信号途径的外层，该酶显然具有底物的初始对接位置。？-分泌酶是一个新兴的膜内蛋白水解酶家族的创始成员，该家族的活性部位显然嵌入在脂质双层中。尽管取得了令人瞩目的进展，但关于这种不同寻常的重要蛋白酶仍有许多未知之处。为了促进对β-分泌酶的机制的了解，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)分泌酶活性部位的性质是什么？底物对接部位？(2)-分泌酶如何识别和处理其底物？(3)？-分泌酶如何变构调节以阻止APP的蛋白分解而不是Notch？这个项目的目标是了解分泌酶，这是一种复杂的酶，对阿尔茨海默病的病因至关重要。有机小分子将被用来作为探针来表征这种酶，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.