High-throughput compound screening for modulators of insulin-degrading enzyme.

描述（由申请人提供）：降解淀粉样蛋白（A）的蛋白酶，其在阿尔茨海默病（AD）中异常积累，已成为体内淀粉样蛋白生成的关键调节剂，但仅开始探索其治疗潜力。胰岛素降解酶（IDE）是一种重要的A β-降解蛋白酶，近年来在实验、遗传和动物模型研究中得到了广泛的应用。重要的是，最近的证据表明IDE活性可能通过几种机制中的任何一种来增加，包括内源性抑制剂的置换和其分泌到细胞外空间的调节。此外，IDE的新晶体结构表明，这种蛋白酶具有非正统的酶学性质，可以利用直接激活蛋白酶多达40倍。在这里，我们提出使用针对IDE激活剂的检测优化的基于细胞的测定对约550，000种化合物的化学多样性文库进行超高通量筛选（uHTS）。主要试验的开发和实施将主要由Scripps佛罗里达经验丰富的uHTS中心以适度的成本进行，从而可以将更多的注意力集中在鉴定真正的IDE激活剂并表征其作用机制所必需的关键次要试验上。我们的长期目标是确定适用于培养细胞和体内的药效团，这可能会导致开发新的疗法来治疗这种毁灭性的疾病。公共卫生安全：该提案的目标是测试大量分子的潜力，以影响已知调节阿尔茨海默病的基本生物过程，特别是与胰岛素降解酶有关的生物过程。发现的分子将被评估其可能的治疗潜力，并有可能通过未来的资助提案开发成新药。

DESCRIPTION (provided by applicant): Proteases that degrade the amyloid ¿-protein (A¿), which accumulates abnormally in Alzheimer's disease (AD), have emerged as critical regulators of amyloidogenesis in vivo, yet have only begun to be explored for their therapeutic potential. Accumulating experimental, genetic and animal modeling studies implicate insulin-degrading enzyme (IDE), as a particularly important A¿-degrading protease. Importantly, recent evidence shows how IDE activity might be increased by any of several mechanisms, including the displacement of endogenous inhibitors and modulation of its secretion into the extracellular space. Moreover, new crystal structures of IDE show that this protease possesses unorthodox enzymological properties that can be exploited to directly activate the protease as much as 40-fold. Here we propose to conduct ultra high- throughput screening (uHTS) on a chemically diverse library of ~550,000 compounds using a cell-based assay optimized for the detection of IDE activators. The development and implementation of the primary assay will largely be performed by Scripps Florida's highly experienced uHTS Core for a modest cost, permitting greater attention to be focused on critical secondary assays essential for identifying bone fide IDE activators and characterizing their mechanism(s) of action. Our long-term goal is to identify pharmacophores suitable for use in cultured cells and in vivo, which may lead to the development of novel therapies to treat this devastating disease. PUBLIC HEALTH RELELVANCE: The goal of this proposal is to test a large collection of molecules for their potential to affect fundamental biological processes known to regulate Alzheimer's disease, specifically relating to insulin-degrading enzyme. Discovered molecules will be evaluated for their possible therapeutic potential, and potentially developed into novel drugs through future funding proposals.