Targeting ApoE4 as a Therapeutic Strategy for Alzheimer's Disease

描述(申请人提供)：载脂蛋白(Apo)E4是阿尔茨海默病(AD)的主要遗传风险因素，而apoE4携带者占AD病例的65%-80%。载脂蛋白E4增加了AD的发生，降低了AD的发病年龄，大量证据表明，ApoE4在AD的神经退行性变和病理生理中起着基础性作用。我们发现apoE4由于其独特的结构特征被称为apoE4结构域相互作用，非常容易被神经元中的蛋白水解性切割，产生神经毒性片段。主要的有毒片段apoE4(1-272)在AD患者的脑和脑脊液中的水平比非痴呆患者和在神经元中表达人apoE4的转基因小鼠高得多。这些转基因小鼠有学习和记忆障碍以及神经病理，包括突触-树突连接的丧失，这与学习障碍的发生有关。ApoE4和apoE4(1-272)的毒性是通过它们抑制线粒体功能而介导的，线粒体功能是导致AD神经变性和认知功能障碍的初始事件。因此，许多研究证实apoE4是AD药物的极佳靶点。我们发现了与apoE4结合的小分子来修改蛋白质的结构，以减少结构域相互作用和蛋白水解性切割，以防止其毒性。我们的领先apoE4结构校正器(ApoE4SC)PY-101可以保护神经元免受apoE4诱导的线粒体毒性，并逆转体外培养的神经突起生长的损害。在体内，PY-101具有良好的药代动力学(PK)和脑生物利用度，阻止神经元特异性烯醇化酶(NSE)-apoE4转基因小鼠脑内有毒apoE4(1-272)的形成，并保护脑内线粒体损伤，这是AD疾病进展的初始事件。在这项U01拨款中，我们建议进行临床前研究，以评估PY-101作为治疗AD的唯一药物的疗效。我们将在NSE-apoE4和APP/apoE4 AD动物模型上测试PY-101对认知障碍和神经病理的阻断作用，以验证PY-101作为治疗AD的药物的有效性。此外，我们还将根据PY-101周围的构效关系，利用我们的药效团模型对apoE4SCs的性质进行优化。我们将使用这个模型来围绕一个新的化学系列建立一个化学优化程序，以提高我们的原型apoE4SC的效力，同时保持良好的PK和高大脑渗透率。利用该模型、药物化学和我们的高通量筛选试验，我们将优化我们的apoE4SCs的疗效和药学特性。为此，将根据我们主要的GFP-apoE4-eDHFR FRET实验筛选小分子，该实验测量靶标结合和破坏apoE4结构域相互作用的能力，以增加apoE4的稳定性。优化的apoE4SCs将接受稳定性、PK和药效学研究，以选择用于测试AD小鼠模型疗效的线索。牵头的apoE4SC将接受标准研究新药的临床前开发，目标是启动临床研究，以测试治疗D的安全性和有效性，并进入IND。 与公共卫生相关：阿尔茨海默病是一种毁灭性的神经退行性疾病，在美国影响着500多万人，每年给医疗保健社区造成1720亿美元的损失。我们建议找出针对载脂蛋白E4的新的小分子，旨在中和该蛋白及其片段对线粒体的毒性，以防止阿尔茨海默病的神经退化和进展。在这些研究中发现的有效化合物将在临床前研究中开发，目的是在人类身上测试选定的临床候选药物在治疗阿尔茨海默病方面的安全性和有效性。

DESCRIPTION (provided by applicant): Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD), and apoE4 carriers account for 65-80% of AD cases. ApoE4 increases the occurrence and lowers the age of onset of AD, and considerable evidence suggests that it has a fundamental role in the neurodegeneration and pathophysiology of AD. We showed that apoE4, because of its unique structural feature referred to as apoE4 domain interaction, is highly susceptible to proteolytic cleavage in neurons, generating neurotoxic fragments. The primary toxic fragment, apoE4(1-272), is found at much higher levels in the brain and cerebrospinal fluid of AD patients than non-demented individuals and in transgenic mice expressing human apoE4 in neurons. These transgenic mice have learning and memory impairments and neuropathology, including loss of synaptodendritic connections, which correlates with the onset of learning deficits. The toxicity induced by apoE4 and apoE4(1-272) is mediated by their inhibition of mitochondrial function, an initial event leading to neurodegeneration and cognitive impairment in AD. Thus, a number of studies have validated apoE4 as an excellent target for AD drugs. We discovered small molecules that bind apoE4 to modify the protein's structure to reduce domain interaction and proteolytic cleavage to prevent its toxicity. Our lead apoE4 structure corrector (apoE4SC), PY-101, protects neurons from apoE4-induced mitochondrial toxicity and reverses the impairment of neurite outgrowth in vitro. In vivo, PY-101 has good pharmacokinetics (PK) and brain bioavailability, blocks formation of toxic apoE4(1-272) in neuron-specific enolase (NSE)-apoE4 transgenic mouse brain, and protects against mitochondrial impairment in brain, an initial event in disease progression in AD. In this U01 grant, we propose to conduct the preclinical studies to evaluate the efficacy of PY-101 as a unique drug to treat AD. We will test PY-101 for efficacy in blocking cognitive impairment and neuropathology in the NSE-apoE4 and APP/apoE4 animal models of AD to validate PY-101 as a drug to treat AD. In addition, we will focus on optimizing the properties of apoE4SCs using our pharmacophore model based on the structure-activity relationship around PY-101. We will use this model to build a chemical optimization program around a novel chemical series to improve the potency of our prototypical apoE4SC while maintaining good PK and high brain penetration. Using the model, medicinal chemistry, and our high-throughput screening assays, we will optimize the efficacy and pharmaceutical properties of our apoE4SCs. To do this, small molecules will be screened against our primary GFP-apoE4-eDHFR FRET assay, which measures target engagement and the ability to disrupt apoE4 domain interaction to increase apoE4 stability. Optimized apoE4SCs will be subjected to stability, PK, and pharmacodynamic studies to select leads to test for efficacy in AD mouse models. The lead apoE4SC will be subjected to standard investigational new drug-enabling preclinical development with a goal of initiating clinical studies to test for safety and efficacy in treating D and proceeding to an IND. PUBLIC HEALTH RELEVANCE: Alzheimer's disease is a devastating neurodegenerative disease that affects more than 5 million people in the U.S., and costs the healthcare community $172 billion annually. We propose to identify novel small molecules targeting apolipoprotein E4 that are designed to neutralize the toxicity this protein and its fragments exert on mitochondria to prevent neurodegeneration and progression of Alzheimer's disease. Effective compounds discovered in these studies will be developed in preclinical studies with the goal of testing a selected clinical candidate in humans for safety and efficacy in treating Alzheimer's disease.