Abstract
Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pK(D) = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pK(D) = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest.
Highlights
Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas
The methods applied to fluorescent ligand chemical synthesis have matured significantly and this study has further highlighted the important contribution of the linker to the overall pharmacology of fluorescently labelled G protein-coupled receptors (GPCRs) ligands
We have shown that the non-selective A1AR and A3AR antagonist 3 can be used as the parent ligand to generate higher affinity and subtype-selective fluorescent probes using advantageous amino acid selection within a dipeptide linker
Summary
Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. Fluorescent ligands have been increasingly used in studying GPCRs; for example to determine receptor expression levels in diseased tissues,1 realtime receptor–receptor interactions and signaling,2,3 and as a tracer ligand in a competition binding assay.4 A common approach to the design and synthesis of receptor probes involves tethering a known orthosteric binding moiety to a second ligand or fluorophore via a linker to form a conjugate.5,6 It is desirable to develop general methods that can increase conjugate affinity and selectivity for a target receptor, especially if a receptor subtype-selective pharmacophore is not available.
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