Genetically-Targeted Photo-Pharmacology for Native Opioid Receptors

项目总结 在神经系统中，G蛋白偶联受体(GPCRs)感知神经调节剂启动 细胞内信号级联控制过多的大脑功能。特别重要的是阿片类药物 参与痛觉、奖赏处理和情绪调节的受体。与这些一致 阿片受体是治疗各种疾病的主要药物靶点。Mu-阿片受体激动剂 (MOR)是常见的止痛药，也有潜在的抗抑郁作用。然而，阿片类药物的一个挑战- 基础治疗是指上瘾倾向、耐受性和危险的副作用。不幸的是，限制 在精确的药理学方法中阻碍了我们剖析分子、细胞和 MOR介导的疾病治疗的电路水平机制，并开发改进的治疗策略。 我们之前建立了使用光药理学对GPCRs进行光学控制的方法， 我们将在R61阶段适应MOR。在目标1中，我们将开发一系列新的光开关化合物 对于MOR采用了基于结构预测、化学合成和功能分析相结合的方法。在……里面 目的2，我们将这些化合物改造成可光切换的正交远地拴系配体(PORTL)。 它们通过标记标签(如SNAP、CLIP或Halo)共价连接到靶受体上。要启用目标，请执行以下操作 对于天然受体，我们将进一步扩展我们的系统，开发纳米体-光开关连接物(Npc)，它可以 与天然受体结合并提供PORTL以实现可逆的光学控制。鼻咽癌可以是基因上的- 编码以允许细胞类型靶向或可在体外纯化并以无基因的形式直接应用于样本 接近。总而言之，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 将这一方法推广到不同受体类型的工程框架。 在R33阶段，我们将利用我们在体内的工具箱来探索MOR介导的抗抑郁药物的基础 对小鼠的影响(目标3)。我们假设MORS的激活定位于内侧前额叶的中间神经元 大脑皮质(MPFC)产生去抑制，导致功能障碍的前额叶回路正常化。PORTL和 Npc将能够以足够的时空精度定位到细胞类型和感兴趣的大脑区域。 探讨受体激活与行为调节的关系。我们将使用行为分析 与抑郁相关，以及奖励和依赖的衡量标准，以及预期目标 光激活可以产生抗抑郁作用，副作用最小。为了获得进一步的机械化 洞察力，我们将进行切片电生理和体内双光子钙成像，以衡量 MOR对mPFC活性的激活。这项工作将在体内验证我们的工具包，并提供关键的一步 了解MOR调节治疗抑郁症的机制。

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.