Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

Gazzi, Thais; Brennecke, Benjamin; Atz, Kenneth; Korn, Claudia; Sykes, David A.; Forn-Cuní, Gabriel; Pfaff, Patrick; Sarott, Roman C.; Westphal, Matthias V.; Mostinski, Yelena; Mach, Leonard; Wasinska-Kalwa, Malgorzata; Weise, Marie; Hoare, Bradley L.; Miljuš, Tamara; Mexi, Maira; Roth, Nicolas; Koers, Eline J.; Guba, Wolfgang; Alker, André; Rufer, Arne C.; Kusznir, Eric A.; Huber, Sylwia; Raposo, Catarina; Zirwes, Elisabeth A.; Osterwald, Anja; Pavlovic, Anto; Moes, Svenja; Beck, Jennifer; Nettekoven, Matthias; Benito-Cuesta, Irene; Grande, Teresa; Drawnel, Faye; Widmer, Gabriella; Holzer, Daniela; Wel, Tom van der; Mandhair, Harpreet; Honer, Michael; Fingerle, Jürgen; Scheffel, Joerg; Broichhagen, Johannes; Gawrisch, Klaus; Romero, Julián; Hillard, Cecilia J.; Varga, Zoltán; Stelt, Mario van der; Pacher, Pál; Gertsch, Jürg; Ullmer, Christoph; McCormick, Peter J.; Oddi, Sergio; Spaink, Herman P.; Maccarrone, Mauro; Veprintsev, Dmitry B.; Carriera, Erick; Grether, Uwe; Nazaré, Marc

doi:10.1039/d1sc06659e

Cited by 14 publications

(18 citation statements)

References 45 publications

(51 reference statements)

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“…[6] Cell-permeable fluorescent probes allowed labeling of CBRs in living endogenously expressing cell populations and even animals illustrating the high translational value. [12] Both probes hold potential for studying target occupancy in animal studies or even human trials. [9,48,49] Our focus on the most relevant six ECS protein targets merely scratches the surface regarding the vast possibilities of generating and applying tailor-made reverse design-based chemical probes and illustrating their critical importance for drug discovery.…”

Section: Discussionmentioning

confidence: 99%

“…[41] HU-210 was chosen Nazaré to visualize CB 2 R in cellulo and in vivo. [12,33] Bifunctional probes, which allow attachment of a reporter unit following reaction with its protein target, were used to study DAGL and NAPE-PLD activity in live cells. [34] Reversible and covalent PET tracers were applied to map the catabolic enzymes MAGL and FAAH in mouse and human brain, respectively.…”

Section: Labeled Chemical Probes Ta Rgeting Proteins Of the Ecsmentioning

confidence: 99%

“…Based on a series of 2,5,6-substituted pyridines [47] (Scheme 2), such as 2 having excellent drug-like properties, i.e. high membrane permeability, affinity, potency and selectivity, Nazaré et al reported a series of full agonist pyridine-based fluorescent probes with excellent affinities for both human and mouse CB 2 R. [12] 3, a cell-permeable variant of the probe with a high target specificity was further employed for live cell imaging by super resolution confocal microscopy ranging from native human macrophages to murine splenocytes. This allowed detection and visualization of CB 2 R at the cell membrane but also of intracellular receptor pools in live cells.…”

Section: Labeled Chemical Probes Ta Rgeting Proteins Of the Ecsmentioning

confidence: 99%

“…Incorporation of cell-permeable fluorescent dyes results in small-molecule derived probes capable of visualizing intracellular protein targets, a task which cannot be achieved with traditional antibodies. [12] Their use enables determination of the localization and expression level of targets as well as unravels molecular mechanisms driving pharmacological responses. [13] Techniques such as fluorescence confocal microscopy, flow cytometry and time-resolved Förster resonance energy transfer (TR-FRET) have been successfully applied to study ligand-receptor interactions.…”

Section: Introductionmentioning

confidence: 99%

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Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Guberman

Kosar

Omran

et al. 2022

Chimia

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Labeled chemical probes are of utmost importance to bring drugs from the laboratory through the clinic and ultimately to market. They support and impact all research and discovery phases: target verification and validation; assay development; lead optimization; and biomarker engagement in the context of preclinical studies and human trials. Probes should display high potency and selectivity as well as fulfill specific criteria in connection with absorption, distribution, metabolism, excretion and toxicology (ADMET) profile. Progress in fields such as imaging and proteomics increased the need for specialized probes to support drug discovery. Labeled probes carrying an additional reporter group are valuable tools to meet specific application requirements, but pose significant challenges in design and construction. In the reverse-design approach, small molecules previously optimized in medicinal chemistry programs form the basis for the generation of such high-quality probes. We discuss the reverse design concept for the generation of labeled probes targeting the endocannabinoid system (ECS), a complex lipid signaling network that plays a key role in many human health and disease conditions. The examples highlighted include diverse reporter units for a range of applications. In several cases the reported probes were the product of mutually rewarding and highly cross-fertilizing collaborations among academic and industry research programs, a strategy that can serve as a blueprint for future probe generation efforts.

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Section: Discussionmentioning

confidence: 99%

Section: Labeled Chemical Probes Ta Rgeting Proteins Of the Ecsmentioning

confidence: 99%

Section: Labeled Chemical Probes Ta Rgeting Proteins Of the Ecsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Guberman

Kosar

Omran

et al. 2022

Chimia

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“…As the first GPCR photoaffinity probe, LEI-121 lends itself useful for pharmacological profiling of CB2 ligands via SDS-PAGE and proteomics. In addition, reversible fluorescent CB2 probes with improved spatiotemporal properties have been developed and applied to in vivo zebrafish models. , For the CB1 receptor, a similar probe was developed by Katona and colleagues, which enabled single-molecule receptor visualization in HEK-293 cells using PharmacoSTORM …”

Section: Visualizing and Controlling Lipid Transfer And Actionmentioning

confidence: 99%

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

2022

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Conspectus Signaling lipids, such as the endocannabinoids, play an important role in the brain. They regulate synaptic transmission and control various neurophysiological processes, including pain sensation, appetite, memory formation, stress, and anxiety. Unlike classical neurotransmitters, lipid messengers are produced on demand and degraded by metabolic enzymes to control their lifespan and signaling actions. Chemical biology approaches have become one of the main driving forces to study and unravel the physiological role of lipid messengers in the brain. Here, we review how the development and use of chemical probes has allowed one to study endocannabinoid signaling by (i) inhibiting the biosynthetic and metabolic enzymes; (ii) visualizing the activity of these enzymes; and (iii) controlling the release and transport of the endocannabinoids. Activity-based probes were instrumental to guide the discovery of highly selective and in vivo active inhibitors of the biosynthetic (DAGL, NAPE-PLD) and metabolic (MAGL, FAAH) enzymes of endocannabinoids. These inhibitors allowed one to study the role of these enzymes in animal models of disease. For instance, the DAGL–MAGL axis was shown to control neuroinflammation and the NAPE-PLD–FAAH axis to regulate emotional behavior. Activity-based protein profiling and chemical proteomics were essential to guide the drug discovery and development of compounds targeting MAGL and FAAH, such as ABX-1431 (Lu AG06466) and PF-04457845, respectively. These experimental drugs are now in clinical trials for multiple indications, including multiple sclerosis and post-traumatic stress disorders. Activity-based probes have also been used to visualize the activity of these lipid metabolizing enzymes with high spatial resolution in brain slices, thereby showing the cell type-specific activity of these lipid metabolizing enzymes. The transport, release, and uptake of signaling lipids themselves cannot, however, be captured by activity-based probes in a spatiotemporal controlled manner. Therefore, bio-orthogonal lipids equipped with photoreactive, photoswitchable groups or photocages have been developed. These chemical probes were employed to investigate the protein interaction partners of the endocannabinoids, such as putative membrane transporters, as well as to study the functional cellular responses within milliseconds upon irradiation. Finally, genetically encoded sensors have recently been developed to monitor the real-time release of endocannabinoids with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models. It is anticipated that the combination of chemical probes, highly selective inhibitors, and sensors with advanced (super resolution) imaging modalities, such as PharmacoSTORM and correlative light-electron microscopy, will uncover the fundamental basis of lipid signaling at nanoscale resolution in the brain. Furthermore, chemical biology approaches enable the translation of these fundamental discoveries into clinical solutions for brain dis...

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Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB₂ Receptor

De Paus,

An,

Janssen

et al. 2024

ChemBioChem

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The cannabinoid receptor type 2 (CB2R) is a G protein‐coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI‐121 that stabilized the inactive conformation of the CB2R. Here, we report the structure‐based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click‐chemistry with fluorophore‐azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.

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Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

Abstract: Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent,...

Cited by 14 publications

References 45 publications

Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB₂ Receptor

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Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe

Abstract: Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent,...

Cited by 14 publications

References 45 publications

Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Reverse-Design toward Optimized Labeled Chemical Probes – Examples from the Endocannabinoid System

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB2 Receptor

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Product

Resources

About

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB₂ Receptor