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:
- 金额:$ 19.44万
- 依托单位:
- 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:
- 关键词:ATP Synthesis PathwayAbsence of pain sensationAnalgesicsBehavioralBinding ProteinsBiological AssayBiosensorBrainCNR1 geneCNR2 geneCYP2C9 geneCYP3A4 geneCalciumCalmodulinCannabidiolCannabinoidsCannabisCell membraneCellsChemical EngineeringChemicalsCollaborationsCoupledCouplingCytochrome P450DetectionDimerizationDrug KineticsDrug Side EffectsEndoplasmic ReticulumEngineeringEnsureEnzymesEvolutionFluorescenceFluorescence Resonance Energy TransferGoalsGolgi ApparatusHela CellsHippocampus (Brain)HumanKineticsLaboratoriesLibrariesLigandsLiverMeasuresMemoryMemory impairmentMetabolismMethodsMissionMitochondriaMolecular ConformationMutagenesisNeuronsNoiseOptical reporterOpticsPain managementPeptide Signal SequencesPerformancePerfusionPhage DisplayPharmaceutical PreparationsPharmacologyPhysiologicalProteinsPublic HealthReporterResearchResolutionSignal TransductionSignaling MoleculeSubstance Use DisorderSystemTHC concentrationTechniquesTestingTetrahydrocannabinolTherapeuticTherapeutic EffectTherapeutic UsesTimeTissuesUnited States National Institutes of HealthUniversitiesVariantWashingtonanalogbaseclinical applicationcombinatorialdesigndimerdrug actiondrug distributiondrug metabolismextracellularfluorescence lifetime imagingimprovedin vivoinduced pluripotent stem cellinnovationinterestmitochondrial membranenanobodiesnew technologynoveloxidationphotolysisscaffoldscreeningside effectsingle cell analysissmall moleculespatiotemporalstem cell differentiationtool
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,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:
- 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:
- 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.
- DOI:10.3389/fbioe.2022.844405
- 发表时间:2022
- 期刊:
- 影响因子:5.7
- 作者:Zhang X;Pan Y;Kang S;Gu L
- 通讯作者:Gu L
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。
- DOI:10.1101/2023.09.13.557660
- 发表时间:2023
- 期刊:
- 影响因子:0
- 作者:Ikbal,MdAshif;Kang,Shoukai;Chen,Xiahui;Gu,Liangcai;Wang,Chao
- 通讯作者:Wang,Chao
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Liangcai Gu其他文献
Liangcai Gu的其他文献
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{{ truncateString('Liangcai Gu', 18)}}的其他基金
PIXEL-seq-based spatial, multi-omic profiling for senescent cell mapping with single-cell resolution
基于 PIXEL-seq 的空间多组学分析,用于具有单细胞分辨率的衰老细胞作图
- 批准号:
10907054 - 财政年份:2021
- 资助金额:
$ 19.44万 - 项目类别:
PIXEL-seq-based spatial, multi-omic profiling for senescent cell mapping with single-cell resolution
基于 PIXEL-seq 的空间多组学分析,用于具有单细胞分辨率的衰老细胞作图
- 批准号:
10494128 - 财政年份:2021
- 资助金额:
$ 19.44万 - 项目类别:
PIXEL-seq-based spatial, multi-omic profiling for senescent cell mapping with single-cell resolution
基于 PIXEL-seq 的空间多组学分析,用于具有单细胞分辨率的衰老细胞作图
- 批准号:
10375968 - 财政年份:2021
- 资助金额:
$ 19.44万 - 项目类别:
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:
10040050 - 财政年份:2021
- 资助金额:
$ 19.44万 - 项目类别:
De Novo Engineering of Small Molecule-Actuatable Biosensors for Cell Therapy
用于细胞治疗的小分子可驱动生物传感器的从头工程
- 批准号:
9752608 - 财政年份:2018
- 资助金额:
$ 19.44万 - 项目类别:
De Novo Engineering of Small Molecule-Actuatable Biosensors for Cell Therapy
用于细胞治疗的小分子可驱动生物传感器的从头工程
- 批准号:
10222721 - 财政年份:2018
- 资助金额:
$ 19.44万 - 项目类别:
De Novo Engineering of Small Molecule-Actuatable Biosensors for Cell Therapy
用于细胞治疗的小分子可驱动生物传感器的从头工程
- 批准号:
10461083 - 财政年份:2018
- 资助金额:
$ 19.44万 - 项目类别: