Development of a HTS assay for modulators of presenilin 1 conformation

描述（由申请人提供）：据报道，淀粉样蛋白42/40比例增加与大脑老化和阿尔茨海默病（AD）有关，并与记忆丧失和AD发病机制有关。通过淀粉样蛋白前体蛋白（APP）的裂解产生Ass的最后酶促步骤是由早老素1 （PS1）依赖的？分泌酶复杂。PS1/？因此，分泌酶的裂解决定了将产生哪种A¿物种：Ab40或高纤维原性和神经毒性的A¿42。重要的是，超过150个已知的PS1突变，分散在整个分子中，都导致常染色体显性家族性AD (FAD)，并增强A¿42代。因此，调节A¿42/40比值具有特殊的治疗重要性。来自几个实验室以及我们自己的最新数据支持PS1构象的细微变化可能会改变？-分泌酶在APP上的裂解位点，从而影响A¿42/40比值。由于PS1构象与A¿42/40比值相关，因此发现变构调节PS1/g-分泌酶的化合物，而不是抑制？分泌酶功能，将提供一个有吸引力的治疗策略。此外，2010年2月在亚利桑那州凤凰城举行的会议讨论了阿尔茨海默病预防倡议（API）。这个想法是开始在那些即将面临阿尔茨海默病风险但没有症状的人身上测试候选药物。这些是FAD PS1 E280A突变携带者，来自哥伦比亚一个大家庭。然而，令人担忧的是，一些FAD PS1突变可能对特定药物产生耐药性（Czirr et al., 2008）。因此，有一个试验将允许筛选PS1/？在不抑制其功能的情况下，测试先导化合物的效力/剂量依赖性/毒性等对于开发基于fad的治疗方法至关重要。我们在完整细胞中使用一种新的基于FRET的技术，观察结果支持一个具有以下重要可测试特征的模型：FAD PS1突变使PS1 N-末端（NT）和环域靠近在一起，这表明由于PS1/？-分泌酶活性位点与APP底物。我们最近开发了一种新的分子报告探针GFP-PS1-RFP (G-PS1-R)，它可以监测完整细胞和/或活细胞中的分子内相互作用。我们使用逐细胞显微镜方法的初步数据显示，影响A¿42/40比率的遗传和药理学操作始终改变G-PS1-R构象（nt环接近性）。我们建议将这种基于fret的检测配置为高通量格式，以1)测试许多FAD PS1突变改变PS1构象的能力，以及2)使用该检测筛选能够“纠正”与a¿42/40比值升高相关的异常致病性PS1构象的变构调制剂的分子文库。如果成功，该项目将有助于确定候选治疗线索并评估其疗效，从而为预防或治愈AD的新治疗策略提供原理验证概念，甚至可能减缓衰老的一些有害影响。

DESCRIPTION (provided by applicant): Increased amyloid ¿ (A¿) 42/40 ratio has been reported in aging brain and in Alzheimer's disease (AD), and is linked to memory loss and AD pathogenesis. The final enzymatic step in generating Ass via cleavage of the amyloid precursor protein (APP) is performed by the presenilin 1 (PS1) dependent ?-secretase complex. The precision of the PS1/?-secretase cleavage, thus, determines which A¿ species will be produced: Ab40 or highly fibrillogenic and neurotoxic A¿ 42. Importantly, over 150 known mutations in PS1, scattered throughout the molecule, all lead to autosomal dominant familial AD (FAD) and enhance A¿ 42 generation. Thus, regulation of the A¿ 42/40 ratio are of particular therapeutic importance. Recent data from several laboratories, as well as our own, support the possibility that subtle changes in PS1 conformation shift the ?-secretase cleavage site on APP, and thus affect the A¿ 42/40 ratio. Since PS1 conformation correlates with the A¿ 42/40 ratio, finding compounds that allosterically modulate PS1/g-secretase, as oppose to inhibit ?-secretase function, would provide an attractive therapeutic strategy. Moreover, February, 2010 meeting in Phoenix, AZ, discussed the Alzheimer's Prevention Initiative (API). The idea is to start testing candidate drugs in people who are at imminent risk for Alzheimer disease but have no symptoms. These are FAD PS1 E280A mutation carriers, members of a large Colombian family. The concern is, however, that some FAD PS1 mutations could be resistant to a particular drug (Czirr et al., 2008). Thus, having an assay that will allow to screen for allosteric modulators of PS1/?-secretase without inhibiting its function, and to test the potency/dose dependence/toxicity etc. of a lead compound would be crucial for development of FAD-based therapeutics. Our observations, using a novel FRET based techniques in intact cells support a model with the following important, testable features: FAD PS1 mutations bring PS1 N- terminus (NT) and loop domain close together, and this represents a pathogenic change in conformation associated with increased A242 production due to altered alignment of the PS1/?-secretase active site with APP substrate. We have recently developed a novel molecular reporter probe, GFP-PS1-RFP (G-PS1-R), which allows monitoring intra-molecular interactions in intact and/or live cells. Our preliminary data using cell-by-cell microscopy approaches show that both genetic and pharmacological manipulations that affect the A¿ 42/40 ratio consistently change G-PS1-R conformation (NT-loop proximity). We propose to configure this FRET-based assay to a high-throughput format to 1) test numerous FAD PS1 mutations for their ability to alter PS1 conformation, and 2) to use this assay to screen Molecular Libraries for allosteric modulators able to "correct" abnormal pathogenic PS1 conformation associated with elevated A¿ 42/40 ratio. If successful, this project will help to identify candidate therapeutic leads and evaluate their efficacy, thus providing a proof-of-principle concept for novel therapeutic strategy to prevent or cure AD, which may even slow down some deleterious effects of aging. PUBLIC HEALTH RELEVANCE: Due to the increased average population age, and thus increased number of affected individuals and associated tremendous healthcare costs, AD has become an urgent public health concern in the U.S. The unmet medical need for developing novel and effective AD therapeutics has been recognized by NIH. We propose to develop and validate an HTS assay to monitor PS1 conformation in intact/live cells. This assay will be used to identify compounds, allosteric modulators of the PS1/?-secretase able to "correct" abnormal pathogenic conformation associated with increased generation of the neurotoxic A242 species. If successful, this project will help to identify candidate therapeutic leads and evaluate their efficacy, thus providing a proof-of-principle concept for novel therapeutic strategy to prevent or cure AD, which may even slow down some deleterious effects of aging.