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

描述（由申请人提供）：目前还没有治疗ALS的方法，并且它是普遍致命的。该提案中的研究将确定可以使用包含物形成作为主要读数来抑制TDP-43聚集的化合物。蛋白质聚集已被认为是多种神经退行性疾病的主要驱动力。TDP-43是ALS药物治疗的最有前途的靶点之一，因为它是肌萎缩侧索硬化症（ALS）中作为包涵体积累的主要蛋白质之一，它显示出强烈的聚集倾向，TDP-43中的突变引起家族性ALS，并且我们观察到突变增加TDP-43形成由聚集蛋白组成的包涵体的倾向。我们进行了化学筛选，以确定抑制TDP-43聚集的小分子，使用高通量资源的药物发现实验室在神经变性（LDDN）在布里格姆妇女医院。本提案中的工作描述了二级筛选和药物化学，以测试和优化包含抑制和神经保护。该提案中使用的优化研究将主要使用皮质和运动神经的原代培养物， 表达人TDP-43的神经元（WT和A315 T）。一些研究还将使用可诱导表达WT TDP-43的神经元PC 12细胞。在每一种情况下，发展细胞质TDP-43夹杂物。我们还将使用C.表达TDP-43的线虫。我们筛选了75，000种新型化合物和1600种FDA批准的生物活性化合物，在10种不同的化学支架上鉴定了22种抑制包合物形成的先导化合物。本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。这项工作将在波士顿Wolozin博士的实验室通过反复的生物测试过程来完成 大学医学院，并通过药物化学的化学优化，在LDDN进行。目的一是对高通量荧光包结法筛选的先导化合物进行验证。我们将从筛选活动中获得22种先导化合物，并测试每种化合物在TDP-43介导的毒性的神经元模型中抑制变性和包涵体形成的能力。我们将研究皮层和运动神经元的原代培养。我们还将研究C的行。表达WT或A343 T TDP-43的线虫，以研究行为和包涵体形成。目标2将侧重于化学优化。我们将使用药物化学来优化两种最佳先导化合物的药理学特性。我们将使用一个迭代的过程，其中的铅化合物进行化学修饰，包合物分散测试。

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.