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.

项目摘要：在我作为科学家的职业生涯中，我打算做出的众多贡献之一就是发现 和/或开发可以用作参与人类疾病的膜蛋白的化学问题的化合物。在 该提案的背景，感兴趣的疾病是阿尔茨海默氏病（AD），我打算关注的蛋白质靶标 淀粉样前体蛋白（APP）。广告是美国第六个主要死因，影响三分之一的人 65岁，据估计，到2050年，全国损失了1.1万亿美元的医疗保健。 对于淀粉样蛋白前体蛋白，是第一个描述的遗传突变体，导致常染色体显性剂 家族广告（FAD；也称为早期AD）。淀粉样蛋白级联假设证明了分子 AD肽的积累促进了AD和痴呆的标志，Aβ肽是APP蛋白水解的产物 通过β-分泌酶清洁酶和与AD相关的praquenige斑块的主要成分之后，通过γ-分泌酶。 来自PSEN1和PSEN2中显性突变的发现的进一步遗传证据，这些突变是催化的代码 γ-分泌酶复合物的蛋白质亚基支持淀粉样蛋白生成途径在AD中的参与。临床 某些早期以Aβ为中心的治疗（例如γ-分泌酶抑制剂[GSIS]）的失败至少在 一部分，对复合物的广谱抑制作用的脱靶效应。尚未实现的药物目标 彻底探索的是没有可用化学问题的APP的C99域，它是直接的先驱 Aβ，已显示出参与AD发病机理的几个方面。基础科学问题该提案的目的 回答是：我们期望在小型跨膜蛋白之间找到哪些类型的亲和力（KD）（例如C99） 和小分子； C99-s-s-s-sall分子复合物将如何影响伽马 - 分泌酶介导的蛋白水解的蛋白水解 C99？我的驾驶假设是，通过耦合核磁共振（NMR）基于光谱的高通量 筛选（HTS）具有非常仔细的复合验证，我们可以开发出可能有可能的新型小分子 在广泛的疏水环境中，敏捷应用生物学，而没有对其他γ-引入脱靶影响 分泌酶的裂解靶标。更具体地说，我建议筛选专门与C99结合的化合物，从而导致 干扰γ-分泌酶识别其识别，留下其他脱靶泌尿力酶底物易受正常的影响 （健康）乳沟。为了测试这一点，我建议使用NMR和生化测定法验证我的HTS命中率，并使用 药物化学（通过目录和协作通过SAR）创建高亲和力，C99特定的小分子 问题。我还将筛选范德比尔特发现中20,000个化学多样性分子的策划子集 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专门结合应用程序/C99的化合物，因此可以用来证明 淀粉样蛋白生成途径以特定于应用的方式。我的工作将在强大的培训环境中进行 范德比尔特大学的查尔斯·桑德斯的实验室。