Identification of compounds that inhibit aggregation and toxicity of TDP-43

描述(申请人提供)：目前还没有治疗肌萎缩侧索硬化症的方法，它是普遍致命的。这项提案中的研究将识别能够抑制TDP-43聚集的化合物，使用包合形成作为主要读数。蛋白质聚集被认为是多种神经退行性疾病的主要驱动力。TDP-43是肌萎缩侧索硬化症(ALS)中最有希望的药物治疗靶点之一，它是肌萎缩侧索硬化症(ALS)中聚集的主要蛋白质之一，具有强烈的聚集倾向，TDP-43突变导致家族性ALS，我们观察到TDP-43突变增加了TDP-43由聚集蛋白组成的包涵体的倾向。我们利用布里格姆妇女医院神经变性药物发现实验室的高通量资源进行了化学筛选，以确定抑制TDP-43聚集的小分子。本提案中的工作描述了二次筛选和药物化学，以测试和优化包涵体抑制和神经保护。提案中使用的优化研究将主要使用皮质和运动的原代培养 表达人TDP-43(WT和A315T)的神经元。一些研究还将使用诱导表达WT TDP-43的神经元PC12细胞。在每种情况下，细胞胞浆内都有TDP-43包涵体。我们还将利用表达TDP-43的线虫，分析先导化合物在体内预防运动缺陷的有效性。我们筛选了75,000种新化合物和1600种FDA批准的生物活性化合物，并在10种不同的化学支架上鉴定了22种抑制包裹体形成的先导化合物。这项提案中的工作将确定那些在TDP-43神经毒性模型中也提供神经保护的化学物质。这项工作将通过在波士顿沃洛津博士的实验室进行生物测试的迭代过程来完成 在最不发达国家发展网络上进行了大学医学院的研究和通过药物化学进行化学优化。AIM 1将验证高通量荧光包合分析中的先导化合物。我们将从筛选活动中提取我们的22种先导化合物，并在TDP-43介导的毒性神经模型中测试每种化合物抑制退行性变和包涵体形成的能力。我们将研究皮质和运动神经元的原代培养。我们还将研究表达WT或A343T TDP-43的线虫品系，以研究行为和包涵体的形成。目标2将专注于化学优化。我们将利用药物化学来优化这两种最好的先导化合物的药理性质。我们将使用迭代过程，在该过程中，对先导化合物进行化学修饰，测试包裹体分散性。

DESCRIPTION (provided by applicant): There is currently no therapy for ALS and it is universally fatal. The research in this proposal will identify compounds that can inhibit TDP-43 aggregation using inclusion formation as a primary readout. Protein aggregation has been implicated as a primary driving force in multiple neurodegenerative illnesses. TDP-43 is one of the most promising targets for pharmacotherapy of ALS because it is one of the major proteins that accumulate as inclusions in Amyotrophic Lateral Sclerosis (ALS), it shows a strong tendency to aggregate, mutations in TDP-43 cause familial ALS and we observe that mutations increase the tendency of TDP-43 to inclusions composed of aggregated protein. We performed a chemical screen to identify small molecules that inhibit TDP-43 aggregation using the high-throughput resources of the Laboratory of Drug Discovery in Neurodegeneration (LDDN) at Brigham and Women's Hospital. The work in this proposal describes the secondary screens and medicinal chemistry to test for and optimize inclusion inhibition and neuroprotection. The optimization studies used in the proposal will primarily use primary cultures of cortical and motor neurons expressing human TDP-43 (WT and A315T). Some studies will also use neuronal PC12 cells that inducibly express WT TDP-43. In each case, the develop cellular cytoplasmic TDP-43 inclusions. We will also analyze the efficacy of the lead compounds to prevent motor deficits in vivo, using C. elegans expressing TDP-43. Our screen of 75,000 novel compounds and 1600 FDA approved and bioactive compounds identified 22 lead compounds on 10 different chemical scaffolds that inhibit inclusion formation. The work in this proposal will identify those chemicals that also provide neuroprotection in models of TDP-43 neurotoxicity. The work will be done through an iterative process of biological testing in the laboratory of Dr. Wolozin, at Boston University School of Medicine, and chemical optimization via medicinal chemistry, performed at the LDDN. Aim 1 will validate the lead compounds from high-throughput fluorescence inclusion assay. We will take our 22 lead compounds from the screening campaign, and test the ability of each compound to inhibit degeneration and inclusion formation in neuronal models of TDP-43 mediated toxicity. We will examine primary cultures of cortical and motor neurons. We will also examine lines of C. elegans expressing WT or A343T TDP-43, to investigate behavior and inclusion formation. Aim 2 will focus on chemical optimization. We will use medicinal chemistry to optimize the pharmacological properties of the two best lead compounds. We will use an iterative process, in which the lead compounds are chemically modified, tested for inclusion dispersion.