Testing a Novel Approach to Solve the On-target, Off-site Effects of Alzheimer's Drugs

测试一种解决阿尔茨海默病药物的在靶、异位效应的新方法

• 批准号: 9456159
• 负责人: DEBOMOY K LAHIRI
• 金额: $ 23.75万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9456159
• 关键词: Abeta synthesis; Address; Affect; Alzheimer' s Disease; American; Amyloid beta-Protein; Animal Model; Animals; Basic Science; Brain; Buffers; Cell Culture Techniques; Cleaved cell; Complex; Dementia; Development; Disease; Drug Design; Drug Targeting; Ensure; Enzyme-Linked Immunosorbent Assay; Enzymes; Etiology; Event; Financial compensation; Future; Generations; Glycoproteins; Goals; Hepatotoxicity; Human; Kidney; Knock-out; Knockout Mice; Knowledge; Link; Liver; Longevity; Measures; Medical; Medical Research; Methods; MicroRNAs; Modeling; Molecular; Morphologic artifacts; Mus; Neurology; Organ; Organ Specificity; Orthologous Gene; Pharmaceutical Preparations; Pharmacologic Substance; Production; Protein Precursors; Proteins; Proteolysis; RNA; Radiation; Radiation induced damage; Regulation; Risk; Rodent Model; Sampling; Sialyltransferases; Site; Specificity; System; Testing; TimeLine; Tissues; Transgenic Organisms; Translational Research; Translations; Work; abeta toxicity; age related; base; beta-site APP cleaving enzyme 1; drug discovery; experimental study; gamma secretase; human disease; induced pluripotent stem cell; inhibitor/antagonist; innovation; knockout animal; knockout gene; migration; mouse model; novel; novel strategies; oxidative damage; patient safety; pre-clinical research; radiation response; sialylation; small molecule; translation to humans

Alzheimer's disease (AD) affects over 5.4 million Americans. AD is believed to result in whole or in part by accumulation of the toxic amyloid-β peptide (Aβ), making its generation a candidate for treatment. Aβ is generated from the Aβ-precursor protein (APP) via cleavage with β-site APP cleaving enzyme 1 (BACE1), followed by γ-secretase complex cleavage. BACE1 activity is the rate-limiting step in production of Aβ. Recent human trials of BACE1-targeting drugs were discontinued due to liver toxicity. This has been explained by invoking “off-target effects”. We disagree: Such “failed” drugs may have been molecularly “on-target” but acting “off-site” (OnTOS). Specifically for BACE1: In brain, the primary substrate is APP, but in liver, the primary substrate is α-2,6-sialyltransferase (ST6Gal1). ST6Gal1 is an important enzyme in the body's defense against radiation and oxidative damage. Interference in its processing may have produced hepatotoxicity similar to that of the “failed” anti-BACE1 drug trials. Animal model-based tests may miss such risks because they often use transgenic knockout (KO) animals, which may have compensatory mechanisms to lacking BACE1 and potentially important uncharacterized artifacts. These animals also have such short lifespans under sheltered conditions. Radiation and oxidative load will not approach that faced by “wildype (WT) and in the field” humans. Aim 1: Measure levels of BACE1 and orthologs and of APP and ST6Gal1 processing in brain, kidney, and liver samples from BACE1 KO and WT mice treated with vehicle or BACE1 inhibitors (LY2811376 and MK8931) for potential compensating effects in KO vs. drugs. We will use Westerns for total APP and processing of ST6Gal1 and four other BACE1 substrates (distinguished by band migration) and ELISA for soluble APPβ. Impact: Establish empirical multi-organ comparisons to demonstrate OnTOS as an “induced lack” (drug) effect vs. “congenital” (KO) effect. Aim 2: Determine parallels between mouse models and human samples to lay groundwork for human medical studies. We will obtain at least 15 donor-matched samples of human brain, liver and kidney from AD and non-AD subjects. Proteins and miRNA will be measured as in SA1. We will employ western or quantitative ELISA as in 1-1. RNA-enriched extracts to be used for RT-qPCR of different miRNAs. Impact: Any medical research requires translation to humans. The proposed experiments will lay groundwork for such translation. Aim 3: Measure levels of different miRNAs implicated in regulating BACE1 levels. This will provide a frame of reference for future work to take advantage of different endogenous levels of miRNAs to sidestep OnTOS. We will then validate potentially useful miRNA species in primary mouse brain and liver cultures and in iPSC-derived human brain and liver cultures. Impact: The knowledge generated will provide concrete, mechanistic explanations of a pervasive problem in drug design—ignoring OnTOS. It may also suggest a viable alternative to drugs that may target a molecule specifically but offer no organ specificity: Use of miRNAs with levels that innately insulate a “non-target” organ against excess.

阿尔茨海默氏病（AD）影响超过540万美国人。据信广告会全部或部分导致 有毒淀粉样蛋白β肽（Aβ）的积累，使其生成成为治疗的候选者。 Aβ是 通过用β地点应用裂解酶1（BACE1），从Aβ-前体蛋白（APP）产生 然后进行γ-分泌酶复合裂解。 BACE1活性是Aβ产生的限速步骤。最近的 由于肝毒性而停止了对BACE1靶向药物的人体试验。这已经解释了 调用“非目标效果”。我们不同意：这种“失败”药物可能是分子“靶向”，但行动 “异地”（ONTOS）。专门针对BACE1：在大脑中，主要底物是应用，但在肝脏中，主要底物 底物为α-2,6-溶解酶（ST6GAL1）。 ST6GAL1是人体防御的重要酶 辐射和氧化损伤。干扰其加工的干扰可能产生了类似的肝毒性 在“失败”抗BACE1药物试验中。基于动物模型的测试可能会错过此类风险，因为它们经常使用 转基因敲除（KO）动物，可能具有缺乏Bace1的补偿机制 重要的未表征的伪影。这些动物在庇护条件下的寿命也很短。 辐射和氧化负荷不会接近“野生型（WT）和现场”人类所面临的。 目标1：测量BACE1和直系同源物的水平以及APP和ST6GAL1在脑，肾脏和肝脏中的处理 用车辆或BACE1抑制剂（LY2811376和MK8931）处理的BACE1 KO和WT小鼠的样品 KO与药物中的潜在补偿作用。我们将使用西方人进行总应用程序和ST6GAL1的处理 以及其他四个BACE1底物（以频带迁移为特征）和ELISA用于实心APPβ。影响： 建立经验的多器官比较，以表明ONTO作为“诱发的缺乏”（药物）效应与。 “先天性”（KO）效应。目标2：确定鼠标模型和人类样品之间的相似之处 人类医学研究的基础。我们将至少获得15个人脑，肝脏的供体样本 和AD和非AD主题的肾脏。蛋白质和miRNA将如SA1中的测量。我们将雇用 西方或定量ELISA如1-1所示。富含RNA的提取物用于不同miRNA的RT-QPCR。 影响：任何医学研究都需要转化为人类。拟议的实验将为 这样的翻译。 AIM 3：测量在调节BACE1水平的不同miRNA的水平。这会 提供未来工作的参考框架，以利用不同的内源性miRNA 避开安大略省。然后，我们将验证原代小鼠脑和肝培养物中潜在有用的miRNA物种 以及在IPSC衍生的人脑和肝文化中。影响：产生的知识将提供具体， 药物设计中普遍问题的机理解释 - 对安大略省。它也可能暗示一个可行的 替代可能针对分子但没有器官特异性的药物的替代品：使用miRNA 天生将“非目标”器官隔离的水平反对超越。