Genetically-Targeted Photo-Pharmacology for Native Opioid Receptors

天然阿片受体的基因靶向光药理学

• 批准号: 10790584
• 负责人: Joshua Levitz
• 金额: $ 59.84万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10790584
• 关键词: Acute; Agonist; Analgesics; Antibodies; Antidepressive Agents; Behavior; Behavioral; Behavioral Assay; Binding; Biology; Brain; Brain region; Calcium; Cells; Chemicals; Chronic stress; Complex; Coupling; Dangerousness; Darkness; Dependence; Development; Disease; Disinhibition; Drug Addiction; Drug Targeting; Electrophysiology (science); Engineering; Family; Fentanyl; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Image; In Vitro; Interneurons; Label; Ligands; Light; Measures; Medial; Mediating; Mental Depression; Mental disorders; Methodology; Methods; Modification; Molecular; Molecular Probes; Mood Disorders; Moods; Morphine; Mus; N-terminal; Naloxone; Nervous System; Neuromodulator; Neurons; Opioid; Opioid Receptor; Opioid agonist; Optics; Pain management; Pathway interactions; Peptide Signal Sequences; Perception; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Phase; Prefrontal Cortex; Process; Receptor Activation; Rewards; Role; Sampling; Sensory Process; Signal Transduction; Slice; Somatostatin; Specificity; Structure; Synapses; System; Techniques; Testing; Therapeutic; Time; Work; addiction; antagonist; antidepressant effect; azobenzene; beta-arrestin; brain cell; cell type; chemical synthesis; conflict resolution; desensitization; design; expectation; experimental study; improved; in vivo; insight; interest; molecular dynamics; mood regulation; mu opioid receptors; nanobodies; nervous system disorder; neural circuit; neuronal excitability; neuroregulation; pain sensation; pharmacologic; photoactivation; rational design; receptor; receptor internalization; reward processing; side effect; small molecule; spatiotemporal; targeted treatment; temporal measurement; two-photon

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.

项目概要 在神经系统中，G 蛋白偶联受体 (GPCR) 感知神经调节剂以启动 控制大量大脑功能的细胞内信号级联。特别重要的是阿片类药物 有助于疼痛感觉、奖励处理和情绪调节的受体。符合这些 阿片受体作为多种​​疾病的主要药物靶点。 mu-阿片受体激动剂 (MOR) 是常见的镇痛药，也具有作为抗抑郁药的潜力。然而，阿片类药物面临的挑战是 基础治疗是成瘾倾向、耐受性和危险的副作用。不幸的是，限制 药理学方法的精确性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 GPCR 进行光学控制的方法，该方法 我们将在R61阶段适应MOR。在目标 1 中，我们将开发一系列新型光开关化合物 使用基于结构的预测、化学合成和功能分析相结合的 MOR。在 目标 2，我们将改造这些化合物来制造光可切换的正交远程束缚配体（PORTL） 它通过标记标签（例如 SNAP、CLIP 或 Halo）共价连接到目标受体。启用定位 天然受体，我们将进一步扩展我们的系统来开发纳米体-光开关缀合物（NPC）， 与天然受体结合并传递 PORTL 以进行可逆光学控制。 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直接应用于无基因样品中 方法。总之，这将为铁道部提供一个新的、广泛适用的工具集，同时还开发一个 将这种方法扩展到不同受体类型的工程框架。 在 R33 阶段，我们将利用我们的体内工具集来探索 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 MOR 的激活位于内侧前额叶的中间神经元上 皮层（mPFC）诱导去抑制，导致功能失调的前额叶回路正常化。 PORTL 和 NPC 将能够以足够的时空精度瞄准感兴趣的细胞类型和大脑区域 探讨受体激活与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，以及奖励和依赖的措施，以及期望的目标 光激活可以产生抗抑郁作用，且副作用最小。为了获得进一步的机械 洞察力，我们将进行切片电生理学和体内 2 光子钙成像来测量 MOR 激活 mPFC 活动。这项工作将在体内验证我们的工具集，并为实现这一目标迈出关键一步。 了解 MOR 调节抑郁症治疗的机制。