Discovery of small molecules that specifically target the transmembrane C99 domain of the Amyloid Precursor Protein.

发现特异性靶向淀粉样前体蛋白跨膜 C99 结构域的小分子。

• 批准号: 10066062
• 负责人: Manuel Castro
• 金额: $ 3.03万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10066062
• 关键词: Address; Affect; Affinity; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Automobile Driving; Basic Science; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Biology; Catalogs; Cause of Death; Cell membrane; Chemicals; Clinical; Code; Collaborations; Complex; Coupling; Data; Dementia; Development; Disease; Environment; Enzymes; FDA approved; Failure; Future; Gene Proteins; Generations; Genetic; Goals; Hand; Healthcare; Hydrophobicity; In Vitro; Integral Membrane Protein; Kansas; Late Onset Alzheimer Disease; Lead; Length; Libraries; Link; Mediating; Membrane Proteins; Methods; Molecular; Motivation; Multienzyme Complexes; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Nuclear Protein; Pathogenesis; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Presenile Alzheimer Dementia; Process; Property; Protein Subunits; Proteins; Proteolysis; Recombinants; Resistance; Role; Scientist; Specificity; Structure; Structure-Activity Relationship; Testing; Therapeutic; Time; Training; United States; Universities; Validation; Work; abeta accumulation; base; beta secretase; beta-site APP cleaving enzyme 1; career; cost; design; extracellular; familial Alzheimer disease; gamma secretase; high throughput screening; human disease; human old age (65+); inhibitor/antagonist; insight; interest; mutant; nanomolar; notch protein; novel; presenilin-1; presenilin-2; screening; secretase; small molecule; small molecule libraries; success; three dimensional structure

PROJECT SUMMARY: During my career as a scientist, one of the many contributions I intend to make is to discover and/or develop compounds that can be used as chemical probes for membrane proteins involved in human diseases. In the context of this proposal, the disease of interest is Alzheimer’s disease (AD), and the protein target I intend to focus on is the Amyloid Precursor Protein (APP). AD is the 6th leading cause of death in the U.S., affecting one in three people over the age of 65 and is estimated to cost the nation $1.1 trillion in healthcare by the year 2050. The APP gene, which codes for the Amyloid Precursor Protein, was the first described to have genetic mutants that gave rise to autosomal dominant familial AD (fAD; also referred to as early onset AD). The amyloid cascade hypothesis makes the case that the molecular hallmark of AD and ultimately dementia is instigated by the accumulation of Aβ peptides, a product of APP proteolysis by gamma-secretase following β-secretase cleavage and a major component of the presenile plaques associated with AD. Further genetic evidence from the discoveries of dominant mutations in PSEN1 and PSEN2, which code for catalytic protein subunits of the gamma-secretase complex, support the involvement of the amyloidogenic pathway in AD. Clinical failures of the some of the early Aβ-centric therapeutics (such as gamma-secretase inhibitors [GSIs]) were due, at least in part, to the off-target effects of broad spectrum inhibition of the complex. A pharmacological target that has not been thoroughly explored is the C99 domain of APP, which has no available chemical probes, is the immediate precursor to Aβ, and has been shown to involved in several aspects of AD pathogenesis. The basic science questions this proposal aims to answer are: what types of affinities (Kd) can we expect to find between a small transmembrane protein (such as C99) and small-molecules; and how would a C99-small molecule complex affect gamma-secretase-mediated proteolysis of C99? My driving hypothesis is that by coupling nuclear magnetic resonance (NMR) spectroscopy-based high-throughput screening (HTS) with very careful compound validation, we can develop novel small molecules that can potentially perturb APP biology in a broad range of hydrophobic environments without introducing off-target effects on other γ- secretase cleavage targets. More specifically, I propose to screen for a compound that specifically binds to C99, leading to interference with its recognition by γ-secretase, leaving other off-target secretase substrates susceptible to normal (healthy) cleavage. To test this, I propose to validate my HTS hits using both NMR and biochemical assays, and to use medicinal chemistry (via SAR by catalog and by collaboration) to create high-affinity, C99-specific small-molecule probes. I will also screen a curated subset of the 20,000 most chemically diverse molecules in the Vanderbilt Discovery Library to discover new leads. The results from this project will provide fundamental insight into small molecule- membrane protein interactions and provide compounds that specifically bind APP/C99 that can therefore be used to probe the amyloidogenic pathway in an APP-specific manner. My work will be carried out in a robust training environment in the lab of Charles Sanders at Vanderbilt University.

在我的科学家生涯中，我打算做出的许多贡献之一就是发现 和/或开发可用作涉及人类疾病的膜蛋白的化学探针的化合物。在 在这个提议的背景下，感兴趣的疾病是阿尔茨海默病（AD），我打算关注的蛋白质靶点是 淀粉样前体蛋白（APP）。AD是美国第六大死亡原因，每三个人中就有一个 到2050年，预计美国将花费1.1万亿美元用于医疗保健。APP基因编码 淀粉样蛋白前体蛋白，是第一个被描述为具有遗传突变体，引起常染色体显性遗传， 家族性AD（fAD;也称为早发性AD）。淀粉样蛋白级联假说认为， AD和最终痴呆的一个标志是由Aβ肽（APP蛋白水解的产物）的积累引起的 由β-分泌酶裂解后的γ-分泌酶和与AD相关的早老斑的主要成分。 来自PSEN 1和PSEN 2中显性突变的发现的进一步遗传学证据，这些突变编码催化 γ-分泌酶复合物的蛋白亚基支持AD中淀粉样蛋白生成途径的参与。临床 一些早期以Aβ为中心的治疗方法（如γ-分泌酶抑制剂[GSI]）的失败是由于，至少在 部分，以复合物的广谱抑制的脱靶效应。一个药理学靶点， 彻底探索的是APP的C99结构域，它没有可用的化学探针，是直接的前体， Aβ参与AD发病机制的多个方面。基础科学质疑这一提议的目的 回答是：什么类型的亲和力（Kd），我们可以期望找到一个小的跨膜蛋白（如C99） 以及C99-小分子复合物如何影响γ-分泌酶介导的 C99？我的驱动假设是，通过耦合核磁共振（NMR）光谱的高通量 通过非常仔细的化合物验证，我们可以开发出新的小分子， 在广泛的疏水环境中干扰APP生物学，而不会对其他γ- 分泌酶切割靶点。更具体地说，我建议筛选一种特异性结合C99的化合物， 干扰其被γ-分泌酶识别，使其他脱靶分泌酶底物对正常 （健康的）乳沟。为了验证这一点，我建议使用NMR和生化分析来验证我的HTS命中，并使用 药物化学（通过目录SAR和合作），以创建高亲和力，C99特异性小分子 probes.我还将筛选出范德比尔特发现号上2万种化学成分最多样化的分子中的一个精选子集 图书馆发现新线索。该项目的结果将提供对小分子的基本见解- 膜蛋白相互作用，并提供特异性结合APP/C99的化合物， 淀粉样蛋白生成途径。我的工作将在一个强大的培训环境中进行， 范德比尔特大学的查尔斯·桑德斯实验室。