Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor

Singh, Sameek; Oyagawa, Caitlin R M; Macdonald, Courtney; Grimsey, Natasha L.; Glass, Michelle; Vernall, Andrea J.

doi:10.1021/acsmedchemlett.8b00597

Cited by 32 publications

(41 citation statements)

References 26 publications

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“…[20][21][22] Several fluorescent ligands targeting CB2R have been reported. [23][24][25][26][27][28][29][30][31][32][33][34][35] However, in our collective experience the published probes perform less than optimally as judged by at least one of the following criteria: modularity of design for multiple applications, selectivity over CB1R, affinity and specificity for CB2R, photophysical properties, and applicability across species, techniques, and cell types. Furthermore, bifunctional probes that require additional manipulations prior to imaging are often incompatible with live cells.…”

Section: Introductionmentioning

confidence: 99%

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

et al. 2020

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Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent context. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease and to the detection of CB2R in human breast cancer cells.

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Section: Introductionmentioning

confidence: 99%

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

et al. 2020

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“…A cAMP BRET assay showed that the compound acted as an inverse agonist and widefield microscopy imaging showed plasma membrane staining with no detectable intracellular uptake. Furthermore, signal displacement in presence of SR144528 was observed as well [159] …”

Section: Fluorescent Ligands For Gpcrsmentioning

confidence: 84%

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Borgarelli

Klingl

Escamilla-Ayala

et al. 2021

Chemistry A European J

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Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in‐depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target‐tailored small‐molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high‐precision optical techniques to image with an unprecedented resolution at a single‐molecule level, helped unraveling many of the conundrums related to plasma proteins’ life‐cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage‐gated ion channels, ligand‐gated ion channels and G‐coupled protein receptors.

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“…[148] Coupled with three different fluorophores, BODIPY-630/650, BODIPY-FL, and Cy5, the phenolic chromenopyrazole scaffold was utilised for potential CB fluorescent ligands. [125] The C-4 position of the phenyl was found to have better linker tolerance and higher affinity to CB 2 R. Using BODIPY-630/650 as the fluorophore, the binding affinity (pK i (CB 2 R) ¼ 5.80 AE 0.12) was deemed too low to be useful for imaging studies. Extending the PEG linker from 2 to 5 showed no improvement to the fluorophore's imaging potential or selectivity for BODIPY-630/ 650.…”

Section: Fluorescent Classical Cannabinoid Derivativesmentioning

confidence: 99%

Imaging Cannabinoid Receptors: A Brief Collection of Covalent and Fluorescent Probes for CB

Hamilton¹,

Payne²,

Mocerino³

et al. 2021

Aust. J. Chem.

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There has been an expanding public interest towards the notion that modulation of the sophisticated endocannabinoid system can lead to various therapeutic benefits that are yet to be fully explored. In recent years, the drug discovery paradigm in this field has been largely based on the development of selective CB 2 receptor agonists, avoiding the unwanted CB 1 receptor-mediated psychoactive side effects. Mechanistically, target engagement studies are crucial for confirming the ligand-receptor interaction and the subsequent biological cascades that lead to the observed therapeutic effects. Concurrently, imaging techniques for visualisation of cannabinoid receptors are increasingly reported in the literature. Small molecule imaging tools ranging from phytocannabinoids such as tetrahydrocannabinol (THC) and cannabidiol (CBD) to the endocannabinoids as well as the purely synthetic cannabimimetics, have been explored to date with varying degrees of success. This Review will cover currently known photoactivatable, electrophilic, and fluorescent ligands for both the CB 1 and CB 2 receptors. Structural insights from techniques such as ligand-assisted protein structure (LAPS) and the discovery of novel allosteric modulators are significant additions for better understanding of the endocannabinoid system. There has also been a plethora of fluorescent conjugates that have been assessed for their binding to cannabinoid receptors as well as their potential for cellular imaging. More recently, bifunctional probes containing either fluorophores or electrophilic tags are becoming more prevalent in the literature. Collectively, these molecular tools are invaluable in demonstrating target engagement within the human endocannabinoid system.

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Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor

Cited by 32 publications

References 26 publications

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

Imaging Cannabinoid Receptors: A Brief Collection of Covalent and Fluorescent Probes for CB

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