Discovery of novel small molecule antidepressants

严重抑郁障碍是最常见的精神疾病之一，影响着数百万人 世界各地的人。大多数可用于治疗MDD靶向脑通路的药物涉及单胺能 神经递质。尽管采取了这些疗法，多达50%的患者没有好转。 现有的治疗方法。超极化激活的环核苷酸门控(HCN)通道介导Ih，a 电流对控制神经元兴奋性和整合来自其他大脑的突触输入至关重要 地区。HCN受其辅助亚单位TRIP8b(含四肽重复序列的Rab8b-1)的严格调控。 相互作用蛋白)，负责将HCN转运到神经元表面，仅在 大脑。我们的实验室和其他人已经观察到通过其中一个基因缺失而导致的HCN通道功能的丧失 或者通过干扰TRIP8b功能导致抗抑郁药样表型。除了它在大脑中的作用外， HCN对中枢神经系统外的许多功能都是至关重要的，包括心脏的节律形成。 这一关键的HCN功能扰乱了以前在MDD中以HCN为全球目标的努力。我们有 开发了一种新的靶向HCN/TRIP8b的方法来发现潜在的抑郁症治疗药物 与小分子的相互作用。我们已经建立了高通量的荧光偏振(FP) 筛选试验以及二次试验以鉴定HCN/TRIP8b抑制剂。我们已经验证了我们的 通过测试不同的小分子文库进行初步筛选试验，并进一步验证了几个 这些命中在正交分析中确认目标参与度和效力。同时，基于 已知的TRIP8b x射线晶体结构，我们已经在硅胶屏幕上进行了试点，并验证了几种 结果在我们的生化分析中得到了结果。找出效力和疗效更好的抑制剂，并发现 更适合于探头优化的化合物，我们建议进行扩展湿法和硅高法 利用我们建立的一次和二次化验进行吞吐量筛查。在具体目标1中，我们将使用 我们的FP分析测试了一个包含235,000个不同小分子的文库，以了解它们破坏HCN/TRIP8b的能力 有约束力的。我们还将使用我们的In Silico协议来筛选1800万个化合物锌数据库并验证 通过FP分析将所得到的结构作为抑制剂。在特定的目标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试验，包括荧光热移(FTS)、AlphaScreen、 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HITS在一系列基于细胞的分析中的功能活性，包括流式细胞仪， 免疫细胞化学和电生理学。这项建议代表了一种治疗MDD和 我们发现的小分子将广泛用于开发治疗这种未被治疗的疾病的新疗法 医学需要以及探讨HCN通道和TRIP8b在抑郁症中的作用。

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.