adenosine-5--(n-ethylcarboxamide) and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.

Topics: Boron Compounds; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Ligands; Protein Binding; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1

The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.. Chinese hamster ovary cells stably expressing the human adenosine A(1) receptor and a cyclic 3',5' adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [(3)H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5' (N-ethylcarboxamido) adenosine (NECA).. Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A(1) receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.. The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.

Topics: Adenosine-5'-(N-ethylcarboxamide); Alkaline Phosphatase; Animals; Boron Compounds; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dansyl Compounds; Fluorescent Dyes; Genes, Reporter; GPI-Linked Proteins; Humans; Isoenzymes; Ligands; Microscopy, Confocal; Molecular Imaging; Molecular Probe Techniques; Molecular Structure; Radioligand Assay; Receptor, Adenosine A1; Response Elements; Transfection; Xanthines

Fluorescence spectroscopy is becoming a valuable addition to the array of techniques available for scrutinizing ligand-receptor interactions in biological systems. In particular, scanning confocal microscopy and fluorescence correlation spectroscopy (FCS) allow the noninvasive imaging and quantification of these interactions in single living cells. To address the emerging need for fluorescently labeled ligands to support these technologies, we have developed a series of red-emitting agonists for the human adenosine A1-receptor that, collectively, are N6-aminoalkyl derivatives of adenosine or adenosine 5'-N-ethyl carboxamide. The agonists, which incorporate the commercially available fluorophore BODIPY [630/650], retain potent and efficacious agonist activity, as demonstrated by their ability to inhibit cAMP accumulation in chinese hamster ovary cells expressing the human adenosine A1-receptor. Visualization and confirmation of ligand-receptor interactions at the cell membrane were accomplished using confocal microscopy, and their suitability for use in FCS was demonstrated by quantification of agonist binding in small areas of cell membrane.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Boron Compounds; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Fluorescent Dyes; Humans; Ligands; Membrane Microdomains; Microscopy, Confocal; Radioligand Assay; Receptor, Adenosine A1; Spectrometry, Fluorescence; Structure-Activity Relationship