Discovery of novel small molecule antidepressants

新型小分子抗抑郁药的发现

• 批准号: 9263004
• 负责人: Dane M Chetkovich
• 金额: $ 38.63万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-15 至 2017-11-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9263004
• 关键词: Adverse effects; Affect; Antidepressive Agents; Behavior; Behavioral; Binding; Biochemical; Biological; Biological Assay; Brain; Brain region; Cells; Cessation of life; Chemicals; Computer Simulation; Crystallization; Cyclic Nucleotides; Databases; Disease; Disease remission; Electroconvulsive Therapy; Electrophysiology (science); Evaluation; Exhibits; Flow Cytometry; Fluorescence; Fluorescence Polarization; Genes; Genetic; Glutathione S-Transferase; HCN1 gene; Heart; Heart Abnormalities; High Prevalence; In Vitro; Ion Channel; Knock-out; Knockout Mice; Laboratories; Libraries; Major Depressive Disorder; Mediating; Medical; Mental Depression; Mental disorders; Morbidity - disease rate; Mus; Neuraxis; Neurons; Neurotransmitters; Patients; Periodicity; Pharmaceutical Preparations; Pharmacology; Phenotype; Play; Proteins; Protocols documentation; Publishing; Refractory; Roentgen Rays; Role; Series; Structure; Surface; Synapses; Testing; Up-Regulation; Validation; Ventral Tegmental Area; Work; Zinc Compounds; base; brain pathway; cyclic-nucleotide gated ion channels; depressed patient; disability; dopaminergic neuron; experience; experimental study; high throughput screening; immunocytochemistry; improved; inhibitor/antagonist; mortality; neuronal excitability; new therapeutic target; novel; novel strategies; novel therapeutics; response; screening; small molecule; small molecule libraries; social stigma; therapeutic target; trafficking

Major depressive disorder (MDD) is the one of the most common psychiatric diseases, and affects millions of people worldwide. Most drugs available to treat MDD target brain pathways involving monoaminergic neurotransmitters. Despite these therapies, as many as fifty percent of patients do not improve in response to existing therapies. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate Ih, a current that is critical for controlling neuronal excitability and integration of synaptic inputs from other brain regions. HCN is tightly regulated by its auxiliary subunit, TRIP8b (tetratricopeptide repeat-containing Rab8b- interacting protein), which is responsible for trafficking HCN to the neuronal surface and is only expressed in the brain. Our lab and others have observed that loss of HCN channel function through either genetic deletion or by interfering with TRIP8b function leads to an antidepressant-like phenotype. Beyond its role in the brain, HCN is crucial for many functions outside the central nervous system, including rhythmogenesis in the heart. This crucial HCN function has confounded previous efforts to globally target HCN in MDD. We have developed a novel approach to discover new potential depression therapeutics by targeting HCN/TRIP8b interaction with small molecules. We have established a high throughput fluorescence polarization (FP) screening assay as well as secondary assays to identify HCN/TRIP8b inhibitors. We have validated our primary screening assay by testing a diverse library of small molecules and have further validated several of these hits in orthogonal assays to confirm target engagement and potency. Simultaneously, based on the known TRIP8b x-ray crystal structure, we have carried out a pilot in silico screen and verified several of the resulting hits in our biochemical assays. To identify inhibitors with improved potency and efficacy and find compounds more suitable for probe optimization, we propose to carry out expanded wet and in silico high- throughput screening utilizing our established primary and secondary assays. In specific aim 1, we will use our FP assay to test a library of 235,000 diverse small molecules for their ability to disrupt HCN/TRIP8b binding. We will also use our in silico protocols to screen the 18 million compound Zinc database and verify the resulting structures as inhibitors by the FP assay. In specific aim 2, we will assess the potency of our hits using a rational series of in vitro screening assays including fluorescence thermal shift (FTS), AlphaScreen, and glutathione S-transferase (GST) pulldown experiments. Finally, in specific aim 3, we propose to evaluate the functional activity of our hits in a series of cell-based assays including flow cytometry, immunocytochemistry, and electrophysiology. This proposal represents a novel approach to treat MDD and the small molecules that we discover will be broadly useful in developing new therapeutics for this unmet medical need as well as for probing the involvement of HCN channels and TRIP8b in depression.

重度抑郁症（MDD）是最常见的精神疾病之一，影响数百万 全世界的人。大多数可用于治疗涉及单胺能的MDD目标脑途径的药物 神经递质。尽管有这些疗法，但多达百分之五十的患者没有得到改善 对现有疗法。超极化激活的循环核苷酸门控（HCN）通道介导IH，A 电流对于控制其他大脑的突触输入的神经元兴奋性和整合至关重要 地区。 HCN受其辅助亚基Trip8b（四肽重复含Rab8b-- 相互作用的蛋白质），该蛋白负责运输HCN到神经元表面，仅在 大脑。我们的实验室和其他实验室观察到HCN通道函数通过任何一种遗传缺失的丧失 或通过干扰TRIP8B功能会导致抗抑郁样的表型。超越了在大脑中的作用， HCN对于中枢神经系统以外的许多功能至关重要，包括心脏中的节奏发生。 这种至关重要的HCN功能使以前在MDD中全球靶向HCN的努力混淆了。我们有 开发了一种新型方法，可以通过靶向HCN/TRIV8B来发现新的潜在抑郁疗法 与小分子的相互作用。我们已经建立了高通量荧光极化（FP） 筛选测定和辅助测定法以鉴定HCN/TRIP8B抑制剂。我们已经验证了我们的 通过测试小分子的多元化库，并进一步验证了几个库来筛选测定法 这些在正交测定中进行的命中以确认目标参与和效力。同时根据 已知的Trip8b X射线晶体结构，我们在计算机屏幕上进行了一个飞行员，并验证了几个 在我们的生化测定中产生的命中。鉴定有效力和功效提高的抑制剂，并找到 化合物更适合于探针优化，我们建议进行湿的湿润，并在硅高 - 利用我们已建立的主要和次要测定的吞吐量筛选。在特定目标1中，我们将使用 我们的FP测定法测试了235,000个小分子的库，以破坏HCN/TRIV8B的能力 结合。我们还将使用我们的In In Silico协议来筛选1800万种化合物锌数据库并验证 由FP测定法作为抑制剂所得的结构。在特定的目标2中，我们将评估命中的效力 使用一系列合理的体外筛查测定法，包括荧光热移（FTS），Alphascreen， 和谷胱甘肽S-转移酶（GST）下拉实验。最后，在特定目标3中，我们建议评估 我们命中的功能活性在一系列基于细胞的测定中，包括流式细胞术， 免疫细胞化学和电生理学。该提案代表了治疗MDD和 我们发现的小分子将在开发这种未满足的新疗法方面广泛有用 医疗需求以及探测HCN通道和TRIP8B在抑郁症中的参与。