Barrie Kellam

Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells

G protein‐coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A3 adenosine receptor (A3‐AR) agonist, ABEA‐X‐BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist‐occupied A3‐AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A3‐AR, the fluorescent A3‐AR agonist was able to inhibit forskolin‐stimulated [3H]cAMP production (pEC50=8.57), and this was antagonized by the A3‐selective antagonist MRS1220 (pKB=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC50 = 7.34). Ligand binding to the A3‐AR on the membranes of single cells and subsequent increases in single cell [Ca2+]i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small‐membrane microdomains (~0.2 μm2) revealed two agonist‐occupied A3‐AR components with differing diffusion characteristics (diffusion coefficients=2.65×10−8 and 1.19×10−9 cm2/s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/μm2, respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist‐occupied A3‐receptor complexes, showing different motilities within the membrane of single living cells.—Cordeaux, Y., Briddon, S. J., Alexander, S. P. H., Kellam, B., Hill, S. J. Agonist‐occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells. FASEB J. 22, 850–860 (2008)

Chemical modification of mammalian cell surfaces

The mammalian cell surface is a highly heterogeneous chemical environment with proteins, carbohydrates, lipids and composite molecules controlling vital cell functions. Chemical modification of this environment is a challenge due to the complexity of the surface chemistry and the fragility of the cell. Here, we review recent attempts to perform targeted, non-genetically controlled, changes to cell surface chemistry. Potential applications of cell surface engineering are presented.

Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands

Background and purpose:  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.

Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay

Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A1 and -A3 receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.

Synthesis and in vitro evaluation of lipoamino acid and carbohydrate-modified enkephalins as potential antinociceptive agents

Abstract The principal hindrance to drug uptake into central nervous tissue is the blood–brain barrier (BBB). In addition, potential peptide-based neuropharmaceuticals are rapidly destroyed by intra- and extracellular peptidases. In an attempt to address these biological hurdles, a series of lipo-, glyco- and glycolipo-conjugates of Leu-enkephalin have been synthesised via novel solid-phase strategies, and their in vitro activity assessed using mouse vas deferens (MVD) and guinea pig ileum (GPI) assays. Conjugation of a single lipoamino acid onto the C-terminal of the Leu-enkephalin molecule retains biological activity whilst increasing the molecules overall lipophilicity. Conjugation of a glucuronic acid analogue in an analogous position, however, increases activity 40-fold when compared to the native peptide and induces a high degree of δ -opioid receptor selectivity.

The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs

The past decade has witnessed fluorescently tagged drug molecules gaining significant attraction in their use as pharmacological tools with which to visualize and interrogate receptor targets at the single‐cell level. Additionally, one can generate detailed pharmacological information, such as affinity measurements, down to almost single‐molecule detection limits. The now accepted utilization of fluorescence‐based readouts in high‐throughput/high‐content screening provides further evidence that fluorescent molecules offer a safer and more adaptable substitute to radioligands in molecular pharmacology and drug discovery. One such drug‐target family that has received considerable attention are the GPCRs; this review therefore summarizes the most recent developments in the area of fluorescent ligand design for this important drug target. We assess recently reported fluorescent conjugates by adopting a receptor‐family‐based approach, highlighting some of the strengths and weaknesses of the individual molecules and their subsequent use. This review adds further strength to the arguments that fluorescent ligand design and synthesis requires careful planning and execution; providing examples illustrating that selection of the correct fluorescent dye, linker length/composition and geographic attachment point to the drug scaffold can all influence the ultimate selectivity and potency of the final conjugate when compared with its unlabelled precursor. When optimized appropriately, the resultant fluorescent conjugates have been successfully employed in an array of assay formats, including flow cytometry, fluorescence microscopy, FRET and scanning confocal microscopy. It is clear that fluorescently labelled GPCR ligands remain a developing and dynamic research arena.

Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs‐protein‐coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o‐coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single‐cell ligand‐binding kinetics and the effects of A1‐ and A3‐receptor allosteric modulators on in vivo pharmacology.

Synthesis and Characterization of High-Affinity 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent Ligands for Human β-Adrenoceptors

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of −9.53 and −8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol.1983, 5, 430–437.)

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism

In our previous work, using a fluorescent adenosine‐A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high‐affinity labeling of the active receptor (R∗) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist‐occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645‐occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)‐tagged A3AR exhibited a single diffusing species (0.105 μm2 /s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand‐occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J., Kinetic analysis of antagonist‐occupied adenosine‐A3 receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism. FASEB J. 28, 4211‐4222 (2014). www.fasebj.org

Solid phase synthesis of C-terminal carbohydrate modified enkephalins

1-Azido-2,3,4-tri-O-acetyl-β-D-glucuronic acid (1), immobilised on 2-chlorotrityl and NovaSyn TGR resin, was efficiently reduced to its corresponding resin bound glycosyl amine (3) using propane-1,3-dithiol and triethylamine. Subsequent acylation of (3) with (1), generated the carbohydrate dimer (4). In addition, Fmoc based peptide syntheses performed on (3) and (4) afforded the C-terminal modified Leu and Met-enkephalins (7–10). Preliminary pharmacological evaluation of these compounds identified glycopeptide (7) as a potent and selective δ-opioid receptor agonist.