Development of a HTS assay for modulators of presenilin 1 conformation

描述（由申请人提供）：在老化的大脑和阿尔茨海默病（AD）中已报道淀粉样蛋白（A）42/40比率增加，并且与记忆丧失和AD发病机制有关。通过淀粉样前体蛋白（APP）裂解产生Ass的最后一步酶促步骤由早老素1（PS1）依赖性β-淀粉样前体蛋白（APP）进行。分泌酶复合物PS1/？的精度-因此，分泌酶切割决定了哪种A？物种将被产生：Ab 40或高度纤维化和神经毒性的A？42。重要的是，PS1中超过150个已知的突变，分散在整个分子中，都导致常染色体显性遗传家族性AD（FAD）并增强A42的产生。因此，调节A42/40比率具有特别的治疗重要性。最近的数据从几个实验室，以及我们自己的，支持的可能性，微妙的变化PS1构象改变？APP上的分泌酶切割位点，从而影响A <$42/40比率。由于PS1构象与A42/40比值相关，因此发现了变构调节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）。这个想法是开始测试候选药物的人谁是迫在眉睫的风险阿尔茨海默病，但没有症状。这些是FAD PS1 E280 A突变携带者，一个哥伦比亚大家庭的成员。然而，令人担忧的是，一些FAD PS1突变可能对特定药物具有抗性（Coffee et al.，2008年）。因此，具有将允许筛选PS1/β的变构调节剂的测定，因此，在不抑制其功能的情况下抑制分泌酶，以及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的治疗剂将是至关重要的。我们在完整细胞中使用基于FRET的新技术的观察支持具有以下重要的可测试特征的模型：FAD PS1突变使PS1 N-末端（NT）和环结构域靠近在一起，这代表了与由于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），它允许监测完整和/或活细胞中的分子内相互作用。我们使用细胞显微镜方法的初步数据表明，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环接近度）。我们建议将这种基于FRET的测定配置为高通量形式，以1）测试许多FAD PS1突变改变PS1构象的能力，和2）使用该测定筛选分子文库中能够“校正”与升高的A 42/40比率相关的异常致病性PS1构象的变构调节剂。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，从而为预防或治疗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。 公共卫生相关性：由于平均人口年龄的增加，因此受影响的个体数量增加以及相关的巨大医疗费用，AD已成为美国紧迫的公共卫生问题。NIH已认识到开发新型有效AD治疗剂的未满足的医疗需求。我们建议开发和验证HTS检测，以监测完整/活细胞中的PS1构象。该测定将用于鉴定化合物，PS1/β-受体的变构调节剂。分泌酶能够“纠正”与神经毒性A242物质产生增加相关的异常致病构象。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，从而为预防或治疗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.