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。

基本信息

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
项目类别：
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&apos 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

项目摘要

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：在我作为一名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/或开发可用作人类疾病相关膜蛋白化学探针的化合物。在这个提案的背景下，感兴趣的疾病是阿尔茨海默氏病（AD），而我打算关注的蛋白质目标是淀粉样前体蛋白 (APP) 是美国第六大死因，影响三分之一以上的人。 65 岁，预计到 2050 年，国家医疗保健费用将达到 1.1 万亿美元。APP 基因编码淀粉样前体蛋白，是第一个被描述为具有导致常染色体显性遗传突变的基因突变体家族性 AD（fAD；也称为早发性 AD）淀粉样蛋白级联假说证明了分子因素。 AD 和最终痴呆的标志是由 Aβ 肽（APP 蛋白水解产物）的积累引起的 β-分泌酶裂解后由 γ-分泌酶产生，是与 AD 相关的早老斑的主要成分。 PSEN1 和 PSEN2 显性突变的发现提供了进一步的遗传证据，这些突变编码催化 γ-分泌酶复合物的蛋白质亚基支持淀粉样蛋白生成途径参与 AD 临床。一些早期以 Aβ 为中心的疗法（例如 γ 分泌酶抑制剂 [GSI]）的失败是由于，至少在部分原因是复合物的广谱抑制的脱靶效应尚未确定。彻底探索的是 APP 的 C99 结构域，它没有可用的化学探针，是 APP 的直接前体 Aβ 已被证明参与 AD 发病机制的多个方面。该提案旨在提出基础科学问题。答案是：我们可以期望在小跨膜蛋白（例如 C99）之间找到什么类型的亲和力 (Kd) 和小分子；C99-小分子复合物如何影响γ-分泌酶介导的蛋白水解作用 C99？我的驱动假设是通过耦合基于核磁共振（NMR）光谱的高通量通过非常仔细的化合物验证筛选（HTS），我们可以开发出具有潜力的新型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- 引入脱靶效应更具体地说，我建议筛选一种与 C99 特异性结合的化合物，从而产生干扰γ-分泌酶的识别，使其他脱靶分泌酶底物容易受到正常影响为了测试这一点，我建议使用 NMR 和生化检测来验证我的 HTS 命中，并使用药物化学（通过目录和协作通过 SAR）创建高亲和力、C99 特异性小分子我还将筛选 Vanderbilt Discovery 中 20,000 种化学成分最多样化的分子的精选子集。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，因此可用于探测我的工作将在一个强大的培训环境中进行。范德比尔特大学查尔斯·桑德斯的实验室。