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.

阿尔茨海默病（AD）影响了超过540万美国人。AD被认为是全部或部分由以下原因导致的 毒性淀粉样β肽（Aβ）的积累，使其产生成为治疗的候选者。Aβ是 由Aβ前体蛋白（APP）通过β位点APP裂解酶1（BACE 1）裂解产生， 随后是γ-分泌酶复合物裂解。BACE 1活性是Aβ生成的限速步骤。最近 BACE 1靶向药物的人体试验由于肝毒性而停止。这已经被解释为 引发“脱靶效应”我们不同意：这种“失败”的药物可能在分子上“靶向”，但在药物作用下， “非现场”（OnTOS）。特别是BACE 1：在脑中，主要底物是APP，但在肝中，主要底物是APP。 底物是α-2，6-唾液酸转移酶（ST 6 Gal 1）。ST 6 Gal 1是一种重要的酶，在身体的防御， 辐射和氧化损伤。对其加工过程的干扰可能会产生类似于 “失败的”抗BACE 1药物试验。基于动物模型的测试可能会错过这些风险，因为它们经常使用 转基因敲除（KO）动物，其可能对缺乏BACE 1具有补偿机制， 重要的非特征化伪影。这些动物在庇护条件下的寿命也很短。 辐射和氧化负荷将不会接近“野生型（WT）和在外地”的人所面临的。 目的1：测量脑、肾和肝中BACE 1和直系同源物以及APP和ST 6 Gal 1加工的水平 用溶媒或BACE 1抑制剂（LY 2811376和MK 8931）处理的BACE 1 KO和WT小鼠样品， KO与药物相比的潜在补偿作用。我们将使用Westerns进行总APP和ST 6 Gal处理1 和其他四种BACE 1底物（通过条带迁移区分）以及可溶性APPβ的ELISA。影响： 建立经验性多器官比较，以证明OnTOS作为“诱导缺乏”（药物）效应与 “先天性”（KO）效应。目的2：确定小鼠模型和人类样本之间的相似性 人类医学研究的基础。我们将获得至少15个捐献者匹配的人脑，肝脏， 以及来自AD和非AD受试者的肾脏。蛋白质和miRNA将如SA 1中那样测量。我们会委聘 Western或定量ELISA，如1-1中所述。RNA富集提取物用于不同miRNA的RT-qPCR。 影响：任何医学研究都需要翻译给人类。拟议中的实验将为以下方面奠定基础： 这样的翻译。目的3：测量参与调节BACE 1水平的不同miRNA的水平。这将 为未来的工作提供了一个参考框架，以利用不同的内源性miRNAs水平， 避开OnTOS。然后，我们将在原代小鼠脑和肝脏培养物中验证潜在有用的miRNA种类 以及在iPSC衍生的人脑和肝培养物中。影响：产生的知识将提供具体的， 药物设计中普遍存在的问题的机械解释-忽略OnTOS。这也可能表明一个可行的 可特异性靶向分子但不提供器官特异性的药物的替代品： 使“非目标”器官与过量的药物隔离。