Screening for inhibitors of p300-mediated tau acetylation for Alzheimer's Disease

筛选 p300 介导的 tau 乙酰化抑制剂治疗阿尔茨海默病

基本信息

批准号：
8878152
负责人：
Michelle Arkin
金额：
$ 24.59万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8878152
关键词：
Acetyl Coenzyme A Acetylation Acetyltransferase Affect Affinity Alzheimer&apos s Disease American Antibodies Binding Binding Proteins Biochemical Biological Assay Biological Process Brain Cell Line Cells Chemicals Cherry - dietary Coenzyme A Data Development Diagnostic Disease Dose Drug Targeting Dyes EP300 gene Enzymes Epigenetic Process Evaluation Family member Fluorescence Future Genetic Transcription Grant Health Histone Acetylation Histones Inhibitory Concentration 50 Kinetics Laboratories Lead Length Libraries Literature Malignant Neoplasms Measures Mediating Microtubules Modification Monitor Nerve Degeneration Neurodegenerative Disorders Neurofibrillary Tangles Neurons Outcome Pathogenesis Pathway interactions Patients Peptides Pharmaceutical Chemistry Pharmaceutical Preparations Play Powder dose form Property Protein Biosynthesis Proteins PubChem Publishing Reporting Research Personnel Role Running Screening Result Series Solubility Specificity Staging Surface Plasmon Resonance Tauopathies Technology Therapeutic Time Vesnarinone Work assay development base cofactor compound 30 design drug development drug discovery effective therapy efficacy testing high throughput screening hyperphosphorylated tau in vivo inhibitor/antagonist innovation liquid chromatography mass spectrometry meetings mouse model multicatalytic endopeptidase complex new technology novel p300/CBP-Associated Factor screening small molecule tau Proteins tau aggregation tau expression tau mutation

项目摘要

DESCRIPTION (provided by applicant): The main objective of the proposed project is to develop assays that we will use to discover small-molecule inhibitors of p300, an acetyltransferase enzyme implicated in Alzheimer's disease (AD). We have discovered that p300 acetylates tau in the early stages of AD and developed antibodies that uniquely allow us to probe this activity. We will build a high-throughput screening (HTS) assay that will, for the first time, enable identification of novel compounds that inhibit p300-mediated acetylation of tau. We will also utilize a logical series of assays to validate p300 inhibitors discovered from the HTS and prioritize these compounds for further studies in AD. Tau is a critical protein involved in th early pathogenesis of AD, and its hyperphosphorylated form (p-tau) is a major component of the neurofibrillary tangles (NFTs) found in the brains of AD patients. Evidence also suggests that tau, especially p-tau, causes neurodegeneration in AD. We recently discovered that tau is acetylated (ac-tau) by p300 and that hyperacetylation impairs tau's degradation, resulting in accumulation of pathogenic p-tau. Furthermore inhibiting p300 reduces ac-tau and leads to degradation of tau. We hypothesize that small-molecule inhibitors of p300 will thus clear tau's pathogenic forms and reduce neurodegeneration. Selective drug-like inhibitors of p300 with good in vivo properties are needed to further explore the role of ac-tau in AD. We have shown that C646, an inhibitor of p300, reduces tau acetylation in primary neurons and diminishes p-tau expression after short-term treatment. However, C646's use is limited for in vivo applications, and the few other published p300 inhibitors have low affinity and/or selectivity for this enzyme. Additionally, p300 regulates transcription through histone acetylation, which may play a role in cancer. Selective chemical probes will allow us to dissect the functions of p300 and assess the therapeutic utility of p300 inhibitors. We propose to meet the imperative need for such chemical probes by developing a new HTS assay to measure ac-tau using full-length tau and p300. Ac-tau will be detected by homogenous time-resolved fluorescence (HTRF) using the antibody against ac-tau developed in our lab. To date, no HTS have been published in PubChem for p300, and robust, scalable assays are just beginning to be reported for histone acetylation. In Aim 1, we will develop the HTRF assay for HTS and run a 31,600-compound pilot screen of diverse molecules. In Aim 2, we define a series of assays to prioritize inhibitors based on their mechanisms of action. In addition, we will characterize compounds' selectivity for p300 over related acetyltransferases. Finally, we will determine compounds' inhibition of acetylation in cell lines and primary cortical neurons. Significant and innovative outcomes from this work will be a) development of a suite of assays to screen and thoroughly characterize inhibitors of ac-tau; b) probes of p300 published in PubChem; c) a new HTRF format readily adaptable to other acetyltransferases. Lead compounds will probe the role of ac-tau in AD and other tauopathies, and will be a boon for researchers seeking to understand the biological functions of p300.

描述（由申请人提供）：拟议项目的主要目标是开发检测方法，用于发现 p300 的小分子抑制剂，p300 是一种与阿尔茨海默病 (AD) 相关的乙酰转移酶。我们发现 p300 在 AD 早期阶段乙酰化 tau，并开发了独特的抗体，使我们能够探测这种活性。我们将建立一个高通量筛选（HTS）检测方法，首次时间，能够鉴定抑制 p300 介导的 tau 乙酰化的新化合物。我们还将利用一系列逻辑分析来验证从 HTS 中发现的 p300 抑制剂，并优先考虑这些化合物以用于 AD 的进一步研究。 Tau 是参与 AD 早期发病机制的关键蛋白，其过度磷酸化形式 (p-tau) 是 AD 患者大脑中发现的神经原纤维缠结 (NFT) 的主要组成部分。证据还表明 tau，尤其是 p-tau，会导致 AD 中的神经变性。我们最近发现 tau 被 p300 乙酰化 (ac-tau)，并且过度乙酰化会损害 tau 的降解，导致致病性 p-tau 的积累。此外，抑制 p300 会减少 act-tau 并导致 tau 降解。我们假设 p300 的小分子抑制剂将清除 tau 的致病形式并减少神经退行性变。需要具有良好体内特性的选择性 p300 药物样抑制剂来进一步探索 ac-tau 在 AD 中的作用。我们已经证明，p300 抑制剂 C646 在短期治疗后可减少原代神经元中 tau 乙酰化并减少 p-tau 表达。然而，C646 的用途仅限于体内应用，并且其他少数已发表的 p300 抑制剂对该酶的亲和力和/或选择性较低。此外，p300 通过组蛋白乙酰化调节转录，这可能在癌症中发挥作用。选择性化学探针将使我们能够剖析 p300 的功能并评估 p300 抑制剂的治疗效用。我们建议通过开发一种新的 HTS 测定法来使用全长 tau 和 p300 测量 ac-tau 来满足对此类化学探针的迫切需求。使用我们实验室开发的抗 ac-tau 抗体，通过均质时间分辨荧光 (HTRF) 检测 Act-tau。迄今为止，PubChem 尚未发表 p300 的 HTS，而针对组蛋白乙酰化的稳健、可扩展的检测方法刚刚开始报道。在目标 1 中，我们将开发 HTS 的 HTRF 测定法，并对不同分子进行 31,600 种化合物的试点筛选。在目标 2 中，我们定义了一系列检测方法，根据抑制剂的作用机制对抑制剂进行优先排序。此外，我们将表征化合物对 p300 相对于相关乙酰转移酶的选择性。最后，我们将确定化合物对细胞乙酰化的抑制作用线和初级皮质神经元。这项工作的重大创新成果将是 a) 开发一套筛选和彻底表征 ac-tau 抑制剂的测定方法； b) PubChem 发表的 p300 探针； c) 一种新的 HTRF 形式，易于适应其他乙酰转移酶。先导化合物将探索 act-tau 在 AD 和其他 tau 病中的作用，并且对于寻求了解 p300 生物学功能的研究人员来说将是一个福音。