Genetically Encoded Optical Biosensors for Dissecting Brain Distribution and Metabolism of Cannabinoids

用于解剖大脑分布和大麻素代谢的基因编码光学生物传感器

• 批准号: 10362521
• 负责人: Liangcai Gu
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10362521
• 关键词: ATP Synthesis Pathway; Absence of pain sensation; Analgesics; Behavioral; Binding Proteins; Biological Assay; Biosensor; Brain; CNR1 gene; CNR2 gene; CYP2C9 gene; CYP3A4 gene; Calcium; Calmodulin; Cannabidiol; Cannabinoids; Cannabis; Cell membrane; Cells; Chemical Engineering; Chemicals; Collaborations; Coupled; Coupling; Cytochrome P450; Detection; Dimerization; Drug Kinetics; Drug Side Effects; Endoplasmic Reticulum; Engineering; Ensure; Enzymes; Evolution; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Golgi Apparatus; Hela Cells; Hippocampus (Brain); Human; Kinetics; Laboratories; Libraries; Ligands; Liver; Measures; Memory; Memory impairment; Metabolism; Methods; Mission; Mitochondria; Molecular Conformation; Mutagenesis; Neurons; Noise; Optical reporter; Optics; Pain management; Peptide Signal Sequences; Performance; Perfusion; Phage Display; Pharmaceutical Preparations; Pharmacology; Physiological; Proteins; Public Health; Reporter; Research; Resolution; Signal Transduction; Signaling Molecule; Substance Use Disorder; System; THC concentration; Techniques; Testing; Tetrahydrocannabinol; Therapeutic; Therapeutic Effect; Therapeutic Uses; Time; Tissues; United States National Institutes of Health; Universities; Variant; Washington; analog; base; clinical application; combinatorial; design; dimer; drug action; drug distribution; drug metabolism; extracellular; fluorescence lifetime imaging; improved; in vivo; induced pluripotent stem cell; innovation; interest; mitochondrial membrane; nanobodies; new technology; novel; oxidation; photolysis; scaffold; screening; side effect; single cell analysis; small molecule; spatiotemporal; stem cell differentiation; tool

ABSTRACT Cannabinoids are enjoying a resurgence of interest in potential therapeutic uses, such as pain treatment, but their clinical applications have been hindered due to pronounced psychoactive or other side effects. They can be locally metabolized to active metabolites in the brain and other tissues and it is unknown whether and to what extent they can impact therapeutic and side effects, such as analgesia and memory impairment. The long-term goal is to develop and use new genetically encoded drug and metabolite biosensors to elucidate cannabinoid pharmacology and guide the design of new analgesic therapeutics. The overall objective of this application is to apply new technologies (COMBINES-CID and SMI-seq) to create in vivo biosensors for the major psychoactive cannabis component, Δ9-tetrahydrocannabinol (THC), and its highly potent metabolite, 11-hydroxy-THC, and demonstrate their use in measuring single-cell and subcellular drug distribution and metabolism. The PI’s laboratory recently applied COMBINES-CID to create highly specific chemically induced dimerization systems (CIDs) for cannabidiol (CBD), a structural analog of THC. It is hypothesized that other cannabinoid-selective CIDs can be engineered similarly and then converted to in vivo biosensors, like GCaMP, by coupling to a fluorescence reporter. This hypothesis will be tested by pursuing two specific aims: 1) Generate THC- and 11- hydroxy-THC-induced CIDs with high sensitivity and fast kinetics; and 2) Engineer selected CIDs into fluorescent biosensors and validate their performance by measuring the perfused, photo-uncaged, and metabolized drugs in human iPSC-differentiated neurons. Under the first aim, phage-displayed combinatorial binder libraries constructed with nanobody, monobody, or computationally designed scaffolds will be screened to obtain CIDs with high sensitivity and selectivity (Aim 1A); their detection dynamic ranges will be further optimized by mutagenesis and SMI-seq (Aim 1B). For the second aim, CIDs will be coupled to an optical reporter, such as fluorescence resonance energy transfer or a circularly permuted fluorescent protein, to create the biosensors for fluorescence lifetime imaging (Aim 2A). The biosensors will be genetically encoded and localized to plasma membrane, ER, or Golgi apparatus to measure extracellular and intracellular THC and 11-hydroxy-THC concentrations (Aim 2B) and then study the impact of locally metabolized 11-hydroxy-THC on neuronal mitochondrial ATP synthesis (Aim 2C). The proposed project is innovative in that it will for the first time demonstrate a general solution for creating cannabinoid biosensors. It is significant because obtained biosensors will enable high spatiotemporal analysis of in vivo drug distribution and action. The new method will also have wide use in many other fields by largely expanding the biosensor toolkit for drug, metabolite, and signaling molecule detection.

抽象的 大麻素在潜在的治疗用途（例如疼痛治疗）方面重新引起了人们的兴趣，但是 由于明显的精神活性或其他副作用，它们的临床应用受到阻碍。 在大脑和其他组织中局部代谢为活性代谢物，目前尚不清楚是否以及代谢的结果 它们对治疗和副作用的影响程度，例如镇痛和记忆障碍。 目标是开发和使用新的基因编码药物和代谢物生物传感器来阐明大麻素 药理学并指导新镇痛疗法的设计本申请的总体目标是 应用新技术（COMBINES-CID 和 SMI-seq）为主要精神活性物质创建体内生物传感器 大麻成分 Δ9-四氢大麻酚 (THC) 及其高效代谢物 11-羟基-THC 和 展示它们在测量单细胞和亚细胞药物分布和代谢中的用途。 实验室最近应用 COMBINES-CID 创建高度特异性的化学诱导二聚系统 （CID）代表大麻二酚（CBD），THC 的结构类似物。 CID 可以进行类似的工程设计，然后通过与 该假设将通过追求两个具体目标来检验：1）生成 THC- 和 11- 羟基-THC 诱导的 CID 具有高灵敏度和快速动力学；2) 工程师将 CID 选择为荧光灯； 生物传感器并通过测量灌注、光封闭和代谢药物来验证其性能 在人类 iPSC 分化的神经元中，第一个目标是噬菌体展示组合结合物文库。 将筛选用纳米抗体、单体或计算设计的支架构建的支架以获得 CID 具有高灵敏度和选择性（Aim 1A）；其检测动态范围将进一步优化 诱变和 SMI-seq（目标 1B） 对于第二个目标，CID 将与光学报告基因偶联，例如 荧光共振能量转移或循环排列的荧光蛋白，以创建生物传感器 荧光寿命成像（目标 2A）生物传感器将被基因编码并定位于等离子体。 膜、ER 或高尔基体测量细胞外和细胞内 THC 和 11-羟基-THC 浓度（目标 2B），然后研究局部代谢的 11-羟基-THC 对神经元的影响 线粒体 ATP 合成（目标 2C） 拟议项目的创新之处在于它是首次。 展示了创建大麻素生物传感器的通用解决方案，因为获得的生物传感器具有重要意义。 新方法还将实现体内药物分布和作用的高时空分析。 通过大幅扩展药物、代谢物和信号传导的生物传感器工具包，在许多其他领域得到广泛应用 分子检测。

项目成果

Picomolar-Level Sensing of Cannabidiol by Metal Nanoparticles Functionalized with Chemically Induced Dimerization Binders.

通过化学诱导二聚化粘合剂功能化的金属纳米颗粒对大麻二酚进行皮摩尔水平传感。

• 发表时间: 2023-09-17
• 作者: Ikbal, Md Ashif;Kang, Shoukai;Chen, Xiahui;Gu, Liangcai;Wang, Chao
• 通讯作者: Wang, Chao

Defining molecular glues with a dual-nanobody cannabidiol sensor.

使用双纳米体大麻二酚传感器定义分子胶。

• 发表时间: 2022-02-10
• 影响因子: 16.6
• 作者: Cao, Shiyun;Kang, Shoukai;Mao, Haibin;Yao, Jiayu;Gu, Liangcai;Zheng, Ning
• 通讯作者: Zheng, Ning

Combinatorial Approaches for Efficient Design of Photoswitchable Protein-Protein Interactions as In Vivo Actuators.

作为体内执行器的光可切换蛋白质-蛋白质相互作用的有效设计的组合方法。

• 发表时间: 2022
• 作者: Zhang, Xiao;Pan, Yuxin;Kang, Shoukai;Gu, Liangcai
• 通讯作者: Gu, Liangcai

Polony gels enable amplifiable DNA stamping and spatial transcriptomics of chronic pain.

Polony 凝胶可实现慢性疼痛的可放大 DNA 印记和空间转录组学。

• 发表时间: 2022-11-23
• 影响因子: 64.5
• 作者: Fu, Xiaonan;Sun, Li;Dong, Runze;Chen, Jane Y;Silakit, Runglawan;Condon, Logan F;Lin, Yiing;Lin, Shin;Palmiter, Richard D;Gu, Liangcai
• 通讯作者: Gu, Liangcai