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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

基本信息

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
项目类别：
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

项目摘要

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.

摘要大麻素在潜在的治疗用途中重新引起了人们的兴趣，例如疼痛治疗，但由于明显的精神活性或其它副作用，它们的临床应用受到阻碍。他们可以在大脑和其他组织中局部代谢为活性代谢物，但尚不清楚是否以及如何代谢。在一定程度上，它们可以影响治疗和副作用，如镇痛和记忆障碍。长期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和指导新的镇痛治疗的设计。本应用程序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，以及证明了它们在测量单细胞和亚细胞药物分布和代谢中的用途。PI的一个实验室最近应用COMBINES-CID来创建高度特异性的化学诱导二聚化系统大麻二酚（CBD）是THC的结构类似物。据推测，其他大麻素选择性 CID可以类似地被工程化，然后通过偶联到生物传感器而转化为体内生物传感器，如GCaMP。荧光报告基因。这一假设将通过追求两个具体目标进行测试：1）产生THC-和11- 具有高灵敏度和快速动力学的羟基-THC诱导的CID;和2）将选择的CID工程化为荧光生物传感器，并通过测量灌注，光释放和代谢药物来验证其性能在人类iPSC分化的神经元中。在第一个目标下，噬菌体展示的组合结合剂文库将筛选用纳米抗体、单体抗体或计算设计的支架构建以获得CID 具有高灵敏度和选择性（目标1A）;其检测动态范围将通过以下方式进一步优化：诱变和SMI-seq（目的1B）。对于第二个目的，CID将与光学报告物偶联，例如荧光共振能量转移或环状排列的荧光蛋白，以产生用于荧光寿命成像（Aim 2A）。生物传感器将被基因编码并定位到等离子体中膜、ER或高尔基体，用于测量细胞外和细胞内THC和11-羟基-THC 浓度（目的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（Aim 2C）。该项目的创新之处在于，它将首次展示了制造大麻素生物传感器的通用解决方案。这是有意义的，因为获得的生物传感器将使得能够对体内药物分布和作用进行高时空分析。新方法还将通过大大扩展药物、代谢物和信号传导的生物传感器工具包，在许多其他领域中得到广泛应用分子检测

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Defining molecular glues with a dual-nanobody cannabidiol sensor.

用双纳米型大麻二醇传感器定义分子胶。

DOI：
10.1038/s41467-022-28507-1
发表时间：
2022-02-10
期刊：
Nature communications
影响因子：
16.6
作者：
Cao S;Kang S;Mao H;Yao J;Gu L;Zheng N
通讯作者：
Zheng N

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

DOI：
10.1016/j.cell.2022.10.021
发表时间：
2022-11-23
期刊：
CELL
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

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