Discovery of novel small molecule antidepressants

新型小分子抗抑郁药的发现

基本信息

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
项目类别：
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

项目摘要

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，影响数百万人世界各地的人们。大多数可用于治疗重度抑郁症的药物都针对涉及单胺能的大脑通路神经递质。尽管进行了这些治疗，仍有多达百分之五十的患者的反应没有改善现有疗法。超极化激活的环核苷酸门控 (HCN) 通道介导 Ih，a 对于控制神经元兴奋性和来自其他大脑的突触输入的整合至关重要的电流地区。 HCN 受到其辅助亚基 TRIP8b（含有四肽重复的 Rab8b- 相互作用蛋白），负责将 HCN 运输到神经元表面，并且仅在大脑。我们的实验室和其他人观察到，通过基因删除，HCN 通道功能会丧失或者通过干扰 TRIP8b 功能导致抗抑郁药样表型。除了在大脑中的作用之外， HCN 对于中枢神经系统以外的许多功能至关重要，包括心脏的节律发生。这一关键的 HCN 功能混淆了之前在 MDD 中全球定位 HCN 的努力。我们有开发了一种新方法，通过靶向 HCN/TRIP8b 来发现新的潜在抑郁疗法与小分子的相互作用。我们建立了高通量荧光偏振 (FP) 筛选测定以及鉴定 HCN/TRIP8b 抑制剂的二次测定。我们已经验证了我们的通过测试不同的小分子库进行初步筛选测定，并进一步验证了其中的几个正交测定中的这些命中以确认目标接合和效力。同时，基于已知 TRIP8b X 射线晶体结构，我们在硅屏上进行了试点并验证了其中的几个在我们的生化检测中得到结果。鉴定具有改进效力和功效的抑制剂并发现化合物更适合探针优化，我们建议进行扩展湿法和计算机模拟高利用我们建立的初级和次级测定进行通量筛选。在具体目标 1 中，我们将使用我们的 FP 测定用于测试 235,000 个不同小分子库破坏 HCN/TRIP8b 的能力绑定。我们还将使用我们的计算机协议来筛选 1800 万种化合物锌数据库并验证通过 FP 测定得到的结构作为抑制剂。在具体目标 2 中，我们将评估我们的命中的效力使用一系列合理的体外筛选测定，包括荧光热位移 (FTS)、AlphaScreen、和谷胱甘肽 S-转移酶 (GST) 下拉实验。最后，在具体目标 3 中，我们建议评估我们的命中在一系列基于细胞的测定中的功能活性，包括流式细胞术，免疫细胞化学和电生理学。该提案代表了一种治疗重度抑郁症（MDD）的新方法我们发现的小分子将广泛用于开发针对这一未满足的新疗法医疗需求以及探讨 HCN 通道和 TRIP8b 在抑郁症中的参与。