Targeting ApoE4 as a Therapeutic Strategy for Alzheimer's Disease

描述（由申请人提供）：载脂蛋白（apo） E4是阿尔茨海默病（AD）的主要遗传危险因素，apoE4携带者占AD病例的65-80%。ApoE4增加阿尔茨海默病的发生并降低发病年龄，大量证据表明它在阿尔茨海默病的神经变性和病理生理中起着重要作用。我们发现，apoE4由于其独特的结构特征，即apoE4结构域相互作用，在神经元中极易发生蛋白水解裂解，产生神经毒性片段。原发性毒性片段apoE4（1-272）在阿尔茨海默病患者的大脑和脑脊液中比在非痴呆个体和在神经元中表达人类apoE4的转基因小鼠中含量高得多。这些转基因小鼠有学习和记忆障碍和神经病理学，包括突触树突连接的丧失，这与学习缺陷的发生有关。apoE4和apoE4（1-272）诱导的毒性是通过抑制线粒体功能介导的，线粒体功能是导致AD患者神经变性和认知障碍的初始事件。因此，许多研究已经证实apoE4是AD药物的一个极好的靶点。我们发现结合apoE4的小分子可以改变蛋白质的结构，减少结构域相互作用和蛋白水解裂解，从而防止其毒性。我们的主要apoE4结构纠正剂（apoE4SC） PY-101在体外保护神经元免受apoE4诱导的线粒体毒性，并逆转神经突生长的损伤。在体内，PY-101具有良好的药代动力学（PK）和脑生物利用度，可阻断神经元特异性烯醇化酶(NSE)-apoE4转基因小鼠大脑中毒性apoE4（1-272）的形成，并可防止脑线粒体损伤，这是AD疾病进展的初始事件。在本次U01拨款中，我们建议开展临床前研究，以评估PY-101作为治疗AD的独特药物的疗效。我们将在AD的NSE-apoE4和APP/apoE4动物模型中测试PY-101在阻断认知障碍和神经病理学方面的疗效，以验证PY-101作为治疗AD的药物。此外，我们将重点利用基于PY-101结构-活性关系的药效团模型来优化apoE4SCs的性质。我们将利用该模型构建一个围绕新化学系列的化学优化程序，以提高我们的原型apoE4SC的效力，同时保持良好的PK和高脑穿透性。利用该模型、药物化学和我们的高通量筛选方法，我们将优化apoE4SCs的功效和药物特性。为了做到这一点，小分子将根据我们的主要GFP-apoE4-eDHFR FRET检测进行筛选，该检测测量靶标接合和破坏apoE4结构域相互作用以增加apoE4稳定性的能力。优化后的apoE4SCs将进行稳定性、PK和药效学研究，以选择在AD小鼠模型中测试其功效的引线。先导apoE4SC将接受标准的研究性新药临床前开发，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。

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.