Targeting the mitochondrial pyruvate carrier to treat neurodegenerative disease

描述(申请人提供)：大脑的代谢活动非同寻常，占身体能量需求的近20%。因此，能量代谢不足与多种形式的慢性神经退行性疾病相关或许并不令人惊讶。例如，在阿尔茨海默病(AD)中，患者最早可检测到的缺陷是大脑对葡萄糖的利用减少。流行病学数据加强了糖代谢紊乱之间的联系，因为2型糖尿病是发展为阿尔茨海默病和认知障碍的重要风险因素。事实上，这两种疾病都有几个值得注意的特征，包括胰岛素抵抗(组织无法正确运输和代谢葡萄糖 对胰岛素的反应)、氧化应激、淀粉样变性、认知功能障碍和神经组织萎缩。这种共同的发病机制导致了临床试验，用于治疗2型糖尿病的药物[包括鼻腔注射胰岛素、胰岛素类似物和噻唑烷二酮(TZD)]用于治疗AD和其他神经退行性疾病。不幸的是，这些方法中的每一种都有同样的缺点，限制了它们在糖尿病中的应用，包括转录核受体PPARγ介导的TZD的显著副作用。我们最近报道了TZD具有以前未被发现的多效性效应，其中它们特异性地调节一种重要的代谢物转运体-线粒体丙酮酸载体(MPC，Divakaruni等人)的活性。2013)。MPC将丙酮酸从细胞质运输到线粒体，因此是细胞新陈代谢的关键分支点。TZDS对MPC的轻度抑制可显著刺激人心肌细胞对葡萄糖的摄取，这种作用可被特异性MPC抑制剂UK5099复制。我们已经将我们的工作扩展到神经退行性疾病，提供了有希望的早期数据。低浓度的UK5099还可显著刺激原代培养的大鼠皮质神经元摄取葡萄糖，24小时处理可增强其氧化替代代谢燃料(如酮体)和抵抗兴奋性毒性应激的能力。因此，我们提出了一种治疗AD的新策略：识别温和抑制MPC的类药物化合物，以(I)刺激细胞葡萄糖摄取和(Ii)促进替代燃料的氧化。为了实现这一目标，我们提出如下建议：目标1：通过进一步研究葡萄糖摄取、对兴奋性死亡的抵抗、ROS产生和代谢灵活性，利用13C通量分析来表征原代神经元和星形胶质细胞对轻度MPC抑制的反应。目的2：确定人MPC蛋白复合体和功能重要突变体以及UK5099的高分辨结构，为支持药物开发工作提供详细的框架。目的3：进行化学筛选以确定温和的MPC抑制剂，使用神经元和星形胶质细胞中的一组后续分析来进一步优化潜在的先导化合物。这项关于新型线粒体靶点的多PI项目将生物能量学、药物发现和结构生物学方面的专业知识融合到一个治疗神经退行性疾病的协同计划中。

DESCRIPTION (provided by applicant): The metabolic activity of the brain is extraordinary, accounting for nearly 20% of the body's energy demand. As such, it is perhaps unsurprising that a deficit in energy metabolism is associated with multiple forms of chronic neurodegenerative disease. In Alzheimer's Disease (AD), for example, the earliest detectable defect in patients is diminished glucose utilization by the brain. Epidemiological data reinforces the link between dysregulated glucose metabolism, as type 2 diabetes is a significant risk factor for development of AD and cognitive impairment. In fact, the two diseases share several noteworthy features, including insulin resistance (the inability of tissues to properly transport and metabolize glucose in response to insulin), oxidative stress, amyloidosis, cognitive dysfunction, and atrophy of neural tissues. This shared pathogenesis has led to clinical trials which re-purpose medications used to treat type 2 diabetes [including intranasal insulin, incretin analogues, and thiazolidinediones (TZDs)] to treat AD and other neurodegenerative diseases. Unfortunately, each of these approaches suffer from the same drawbacks that limit their use in diabetes, including significant side effects from TZDs mediated by transcriptional nuclear receptor PPARγ. We recently reported that TZDs have a previously undiscovered, pleiotropic effect in which they specifically modulate the activity of an important metabolite transporter - the mitochondrial pyruvate carrier (MPC, Divakaruni et al. 2013). The MPC transports pyruvate from the cytoplasm into mitochondria, and as such is a crucial branch point in cellular metabolism. Mild inhibition of the MPC by TZDs can acutely stimulate glucose uptake into human myocytes, and this effect can be reproduced by the specific MPC inhibitor UK5099. We have extended our work into neurodegenerative disease with promising early data. A low concentration of UK5099 also acutely stimulates glucose uptake in primary rat cortical neurons, and 24 h treatment enhances their ability to oxidize alternative metabolic fuels (such as ketone bodies) and withstand excitotoxic stress. We therefore propose a novel strategy for the treatment of AD: identify drug-like compounds that mildly inhibit the MPC to (i) stimulate cellular glucose uptake and (ii) promote the oxidation of alternative fuels. To achieve this goal, we propose the following: Aim 1: Characterize the response of primary neurons and astrocytes to mild MPC inhibition with further studies of glucose uptake, resistance to excitotoxic death, ROS production, and metabolic flexibility using 13C flux analysis. Aim 2: Determine the high-resolution structures of the human MPC protein complex and functionally important mutants as well as with UK5099 to provide a detailed framework to support drug development efforts. Aim 3: Conduct a chemical screen to identify mild MPC inhibitors, using a battery of follow-on assays in neurons and astrocytes to further optimize a potential lead compound. This Multi-PI project on a novel mitochondrial target merges expertise in bioenergetics, drug discovery, and structural biology into a synergistic program for the treatment of neurodegenerative disease.