Development of a HTS assay for modulators of presenilin 1 conformation

早老素 1 构象调节剂 HTS 测定的开发

• 批准号: 8050358
• 负责人: OKSANA BEREZOVSKA
• 金额: $ 17.7万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050358
• 关键词: Development; HTS; assay; modulators; presenilin

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.

描述(由申请人提供)：据报道，脑老化和阿尔茨海默病(AD)中淀粉样蛋白(A)42/40比率升高，与记忆丧失和AD发病有关。通过切割淀粉样前体蛋白(APP)产生Ass的最后一步是由依赖于早老素1(PS1)的β-分泌酶复合体执行的。因此，PS1/β-分泌酶切割的精确度决定了将产生哪一种A？种：Ab40或高度纤维素性和神经毒性的A？42。重要的是，散布在整个分子中的150多个PS1已知突变都会导致常染色体显性遗传性家族性AD(FAD)，并增强A？42代。因此，调节A？42/40比率具有特别重要的治疗意义。来自几个实验室的最新数据以及我们自己的数据支持这样一种可能性，即PS1构象的细微变化可能会改变APP上的分泌酶裂解位点，从而影响A？42/40比率。由于PS1构象与A？42/40比率相关，寻找变构调节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氏症预防倡议(API)。这个想法是为了开始在那些面临阿尔茨海默病危险但没有症状的人身上测试候选药物。这些是流行的PS1 E280A突变携带者，来自哥伦比亚的一个大家庭。然而，令人担忧的是，一些流行的PS1突变可能对特定的药物产生抗药性(Chiirr等人，2008年)。因此，有一种方法可以在不抑制PS1/β-分泌酶功能的情况下筛选其变构调节剂，并测试先导化合物的效力/剂量依赖/毒性等，这将是开发基于FAD的治疗药物的关键。我们在完整细胞中使用新的基于FRET的技术观察到的结果支持一个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)和环区紧密结合，这代表了由于PS1/β-分泌酶活性位点与APP底物对齐改变而导致的A242合成增加相关的病理性构象变化。我们最近开发了一种新型的分子报告探针GFP-PS1-RFP(G-PS1-R)，它可以监测完整和/或活细胞中的分子内相互作用。我们使用逐个细胞显微镜方法的初步数据表明，影响A？42/40比率的遗传和药物操作都会一致地改变G-PS1-R构象(NT-环邻近)。我们建议将这种基于FRET的分析配置为高通量形式，以1)测试大量FAD PS1突变改变PS1构象的能力，以及2)使用该方法筛选能够“纠正”与A？42/40升高相关的异常致病PS1构象的变构调节剂的分子文库。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的新治疗策略提供一个原则证明的概念，该策略甚至可能减缓衰老的一些有害影响。 与公共健康相关：由于平均人口年龄的增加，因此受影响的个人数量和相关的巨额医疗成本增加，AD已成为美国一个紧迫的公共卫生问题。开发新的有效AD疗法的医疗需求尚未得到满足，NIH已认识到这一点。我们建议建立和验证一种HTS方法来监测完整/活细胞中的PS1构象。这个测试将被用来鉴定化合物，PS1/？-分泌酶的变构调节剂，能够“纠正”与神经毒性A242物种的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，从而为预防或治愈AD的新治疗策略提供一个原则证明的概念，该策略甚至可能减缓衰老的一些有害影响。