Discovery of Chemical Probes for Psychiatric Disorders and Addiction

摘要 精神疾病和成瘾行为是与适应不良相关的复杂的大脑疾病， 大脑回路的可塑性缺乏足够的平台来快速筛选新的目标， 生理环境严重阻碍了探测器的发现， 导致精神障碍和成瘾的改变，并使未来的治疗设计成为可能。蛋白质- 离子通道复合物内的蛋白质相互作用（PPI）微调神经元的兴奋性， 与精神疾病生物学的联系它们高度特异性和灵活的界面可以使蛋白质- 通道相互作用是探针开发的理想目标。这样的分子探针将提供 神经药理学社区与最佳的研究工具，以解析大脑疾病的复杂性， 最终实现未来的药物设计。 我们已经确定了电压门控Na+（Nav）Nav1.6通道及其辅助调节器之间的PPI 蛋白质，成纤维细胞生长因子14（FGF 14）作为一种新的，功能相关的调节神经元兴奋性， 大脑区域，如皮质-中脑边缘回路，这与情感障碍有关， 认知领域通过成功的基于生物发光的高通量筛选（HTS）， 随后在延髓核（NAc）中的体内研究中，我们发现FGF 14：Nav1.6通道 复合物是糖原合成酶激酶3（GSK 3）通路的一部分，这是一种在细胞内发现异常的信号级联反应， 躁郁症、抑郁症、焦虑症和成瘾症。为了探索FGF 14：Nav1.6复合物的可药用性， 我们采用最小功能域（MFD）方法设计了一个定位于PPI的肽衍生物 界面，并显示其在NAc回路中具有体外到离体活性。这些发现促使我们 开发一种新的管道来识别针对FGF 14：Nav1.6复合物的化学探针， 在皮质-中脑边缘回路中起作用。为了推进针对这个新目标的探测发现活动，我们 设计了一个集成的多模态筛选平台，基于最新的MFD原理， 药理学，包括新设计和验证的生物发光初步筛选试验， 在细胞中重建FGF 14：Nav1.6 C-尾复合物。该流水线还包括必要的计数器， 毒性和无细胞正交试验（目的1），自动膜片钳电生理学作为功能 筛选与构效关系研究和计算机分析相结合（目的2），以及离体 验证NAc电路中选定的探针（目标3）。拟议的管道引入了一个新的，快速的， 集成平台，将加速发现神经元兴奋性的新型化学探针， 为广泛的基于PPI的新型电极导线的治疗前开发提供基础 一系列精神疾病

ABSTRACT Psychiatric diseases and addictive behaviors are complex brain disorders associated with maladaptive plasticity of the brain circuitry. The lack of adequate platforms to rapidly screen against novel targets in physiological environments has significantly hampered probe discovery initiatives that could inform the circuitry alterations leading to mental disorders and addiction and enable the design of future therapeutics. Protein- protein interactions (PPI) within ion channel complexes fine-tune neuronal excitability and are emerging as links to the biology of psychiatric disorders. Their highly specific and flexible interfaces could make protein- channel interactions ideal targets for probe development. Such molecular probes would provide the neuropharmacology community with optimal research tools to parse out brain disease complexities and ultimately enable future drug design. We have identified the PPI between the voltage-gated Na+ (Nav) Nav1.6 channel and its accessory regulator protein, fibroblast growth factor 14 (FGF14) as a novel, functionally relevant regulator of neuronal excitability in brain areas such as the cortico-mesolimbic circuit, which is associated with disorders of the affective and cognitive domains. Through a successful bioluminescence-based high-throughput screen (HTS) and subsequent in vivo studies in the nucleus accumbens (NAc), we discovered that the FGF14:Nav1.6 channel complex is part of the glycogen synthase kinase 3 (GSK3) pathway, a signaling cascade found aberrant in bipolar disorder, depression, anxiety and addiction. To explore the druggability of the FGF14:Nav1.6 complex, we employed a minimal functional domain (MFD) approach to design a peptide-derivative mapped to the PPI interface and showed it has in vitro-to-ex vivo activity in the NAc circuit. These discoveries have prompted us to develop a new pipeline to identify chemical probes against the FGF14:Nav1.6 complex to interrogate its function in the cortico-mesolimbic circuit. To advance a probe discovery campaign against this new target, we have designed an integrated multi-modal screening platform, based on the latest MFD principles of pharmacology that includes a newly designed and validated bioluminescence primary screening assay to reconstitute the FGF14:Nav1.6 C-tail complex in cells. This pipeline also includes the necessary counter, toxicity, and cell-free orthogonal assays (Aim 1), automated patch-clamp electrophysiology as a functional screen in combination with structure-activity relationship efforts and in silico analysis (Aim 2), and ex vivo validation of selected probes in the NAc circuitry (Aim 3). The proposed pipeline introduces a new, rapid and integrated platform that will accelerate the discovery of novel chemical probes for neuronal excitability, providing the foundation for pre-therapeutic development of a new class of PPI-based leads for a broad spectrum of psychiatric disorders.