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

阿尔茨海默病(AD)影响着超过540万美国人。据信，广告的全部或部分原因是 有毒的淀粉样蛋白-β多肽(Aβ)的积累，使其一代成为治疗的候选对象。β是 由Aβ前体蛋白(APP)通过β位点APP裂解酶1(BACE1)切割产生， 其次是γ-分泌酶复合体的切割。BACE1活性是Aβ产生的限速步骤。近期 由于肝脏毒性，BACE1靶向药物的人体试验停止。这一点已经被解释为 调用“偏离目标的效果”。我们不同意：这种“失败”的药物可能是分子上的“靶向”，但作用 “非现场”(Off-Site)。针对BACE1：在大脑，主要底物是APP，但在肝脏，主要底物是APP 底物为α-2，6-唾液酸基转移酶(ST6Gal1)。ST6Gal1是一种重要的酶，在人体防御 辐射和氧化损伤。对其加工过程的干扰可能产生了类似于 “失败”的抗BACE1药物试验。基于动物模型的试验可能会忽略这种风险，因为它们经常使用 转基因基因敲除(KO)动物，可能对缺乏BACE1和潜在的 重要的未确定特征的文物。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)和野外人类所面临的水平。 目标1：测量脑、肾脏和肝脏中BACE1和同源基因以及APP和ST6Gal1加工的水平 从BACE1 KO和WT小鼠中提取的样本经赋形剂或BACE1抑制剂(LY2811376和MK8931)处理后用于 KO与药物的潜在补偿作用。我们将使用西部片进行总APP和ST6Gal1的处理 和其他四种BACE1底物(以条带迁移区分)和ELISA检测可溶性APPβ。影响： 建立经验性的多器官比较，以证明OnTOS是一种“诱导性缺乏”(药物)效应与 “先天”效应。目标2：确定小鼠模型和人类样本之间的相似之处 为人类医学研究奠定基础。我们将获得至少15个捐献者匹配的人脑、肝脏样本 AD患者和非AD患者的肾脏。蛋白质和miRNA将像SA1一样被测量。我们将聘用 Western或定量ELISA法，如1-1。RNA富集物，用于不同miRNAs的RT-qPCR。 影响：任何医学研究都需要翻译成人类。拟议的实验将为以下工作奠定基础 这样的翻译。目的3：检测与调节BACE1水平有关的不同miRNAs的水平。这将是 为今后的工作提供一个参考框架，以利用不同内源水平的miRNAs来 避开OnTOS。然后，我们将在原代培养的小鼠脑和肝脏中验证潜在有用的miRNA物种。 以及在IPSC来源的人脑和肝脏培养中。影响：产生的知识将提供具体的、 对药物设计中普遍存在的问题的机械解释--忽略OnTOS。这也可能暗示着一种可行的 可替代可能针对某个分子但不提供器官特异性的药物：将miRNAs与 使“非靶标”器官不受过量影响的天然水平。

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.