Genetically-Targeted Photo-Pharmacology for Native Opioid Receptors

项目摘要 在神经系统中，G蛋白偶联受体（GPCR）感测神经调质以启动 细胞内信号级联控制着大脑的多种功能。特别重要的是阿片类药物 这些受体有助于疼痛感觉、奖励处理和情绪调节。符合这些 阿片受体是多种疾病的主要药物靶点。μ-阿片受体激动剂 (MOR)是常见的止痛药，也有抗抑郁药的潜力。然而，阿片类药物的挑战- 基于治疗的是成瘾倾向，耐受性和危险的副作用。不幸的是， 药理学方法的精确性阻碍了我们解剖分子、细胞和 回路水平的MOR介导的疾病治疗机制，并开发改进的治疗策略。 我们以前建立了使用药物药理学对GPCR进行光学控制的方法， 我们将在R61阶段适应莫尔。在aim 1中，我们将开发一系列新的光开关化合物 对于莫尔，使用基于结构的预测、化学合成和功能分析的组合。在 目标2，我们将这些化合物改造成光开关正交远程拴系配体（PORTLs） 其用标记标签如SNAP、CLIP或Halo共价连接至靶受体。能够靶向 我们将进一步扩展我们的系统，开发纳米抗体-光开关缀合物（NPC）， 与天然受体结合并递送PORTL用于可逆的光学控制。NPC可以是基因- 编码以允许细胞类型靶向，或者可以在体外纯化并以无基因修饰的方式直接应用于样品。 approach.总之，这将为MORs提供一个新的、广泛适用的工具集，同时还将开发一个 工程框架，用于将这种方法扩展到不同的受体类型。 在R33阶段，我们将利用我们的工具集在体内探索MOR介导的抗抑郁药的基础 对小鼠的影响（目的3）。我们假设MORs的激活定位于内侧前额叶的中间神经元， 皮质（mPFC）诱导去抑制，导致功能失调的前额叶回路正常化。门户网站和 NPC将能够以足够的时空精度靶向感兴趣的细胞类型和大脑区域 探索受体激活和行为调节之间的关系。我们将使用行为分析 与抑郁症的相关性，以及奖励和依赖性的措施与预期的目标， 光活化可以产生抗抑郁效果，副作用最小。为了获得更进一步的机械性 洞察力，我们将进行切片电生理学和体内双光子钙成像，以测量的影响， 莫尔激活对mPFC活性的影响。这项工作将验证我们的工具集在体内，并提供了关键的一步， 了解莫尔调节抑郁症治疗的机制。

PROJECT SUMMARY In the nervous system, G protein-coupled receptors (GPCRs) sense neuromodulators to initiate intracellular signaling cascades that control a plethora of brain functions. Of particular importance are the opioid receptors which contribute to pain sensation, reward processing and mood regulation. Consistent with these roles, opioid receptors serve as major drug targets for a variety of disorders. Agonists of the mu-opioid receptor (MOR) are common analgesics and also have potential as antidepressants. However, a challenge with opioid- based treatment is the propensity for addiction, tolerance and dangerous side effects. Unfortunately, limitations in the precision of pharmacological approaches have hampered our ability to dissect the molecular, cellular and circuit level mechanisms of MOR-mediated disease treatment and develop improved therapeutic strategies. We previously established methodologies for optical control of GPCRs using photopharmacology, which we will adapt for the MOR in the R61 phase. In aim 1, we will develop a range of new photoswitchable compounds for the MOR using a combination of structure-based prediction, chemical synthesis and functional analysis. In aim 2, we will adapt these compounds to make photoswitchable orthogonal remotely-tethered ligands (PORTLs) which covalently attach to target receptors with a labeling tag, such as SNAP, CLIP or Halo. To enable targeting of native receptors, we will further extend our system to develop nanobody-photoswitch conjugates (NPCs) which bind to native receptors and deliver a PORTL for reversible optical control. NPCs can either be genetically- encoded to permit cell type-targeting or can be purified in vitro and directly applied to the sample in a gene-free approach. Together, this will provide a new, widely-applicable toolset for MORs while also developing an engineering framework for extension of this approach across different receptor types. In the R33 phase, we will harness our toolset in vivo to probe the basis of MOR-mediated antidepressant effects in mice (aim 3). We hypothesize that activation of MORs localized on interneurons of the medial prefrontal cortex (mPFC) induce disinhibition that leads to normalization of dysfunctional prefrontal circuits. PORTLs and NPCs will enable targeting to the cell types and brain regions of interest with sufficient spatiotemporal precision to probe the relationship between receptor activation and behavioral modulation. We will use behavioral assays of relevance to depression, as well as measures of reward and dependence with the expectation that targeted photo-activation can produce antidepressant effects with minimal side effects. To gain further mechanistic insight, we will perform slice electrophysiology and in vivo 2-photon calcium imaging to measure the effects of MOR activation on mPFC activity. This work will validate our toolset in vivo and provide a key step toward understanding the mechanism of MOR modulation for depression treatment.