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Objective: Simultaneous activation of β2- and β3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of β1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel β2-and β3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models., Methods: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective β-AR agonist isoprenaline across various rodent β-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis., Results: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis., Conclusions: Our results demonstrate that ATR-127 is a highly effective, novel β2- and β3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TB, BP and ND own stocks in Atrogi AB. ET, AK, ND, SvB, CH, MHB, AS, BP and GY are employed by Atrogi AB. VML is co-founder, CEO and shareholder of HepaPredict AB., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (p K D ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

The histamine H 1 receptor (H 1 R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H 1 R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H 1 R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H 1 R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H 1 R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a , both potent and subtype-selective, enabled H 1 R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H 1 R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

The adenosine A 1 receptor (A 1 AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A 1 AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A 1 AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A 1 AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A 1 AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A 1 AR molecular pharmacology and signaling in living cells.

Organization of G protein-coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A 3 receptor (A 3 AR) to that of the wild-type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA-X-BY630) demonstrated that both wild-type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist-mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A 3 AR and A 3 AR R108A underwent similar agonist-induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit β-arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild-type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist-stimulated β-arrestin recruitment and membrane reorganization of the A 3 AR., (© 2021 Federation of American Societies for Experimental Biology.)

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA-approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome-edited and endogenously expressed GPCRs., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A 2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 μs; D = 30 ± 4 μm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.

Among class A G protein-coupled receptors (GPCR), the human adenosine A 2A receptor (hA 2A AR) remains an attractive drug target. However, translation of A 2A AR ligands into the clinic has proved challenging and an improved understanding of A 2A AR pharmacology could promote development of more efficacious therapies. Subtype-selective fluorescent probes would allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present study, two families of fluorescent probes were designed around the known hA 2A AR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hA 2A AR, selectivity over all other adenosine receptor subtypes and allowed clear visualization of specific receptor localization through confocal imaging. Furthermore, the Alexa Fluor 647-labeled conjugate allowed measurement of ligand binding affinities of unlabeled hA 2A AR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The fluorescent ligands developed here can therefore be applied to a range of fluorescence-based techniques to further interrogate hA 2A AR pharmacology and signaling.

In recent years, several novel aspects of GPCR pharmacology have been described, which are thought to play a role in determining the in vivo efficacy of a compound. Fluorescent ligands have been used to study many of these, which have also required the development of new experimental approaches. Fluorescent ligands offer the potential to use the same fluorescent probe to perform a broad range of experiments, from single-molecule microscopy to in vivo BRET. This review provides an overview of the in vitro use of fluorescent ligands in further understanding emerging pharmacological paradigms within the GPCR field, including ligand-binding kinetics, allosterism and intracellular signalling, along with the use of fluorescent ligands to study physiologically relevant therapeutic agents., (© 2019 The British Pharmacological Society.)

Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A 3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A 3 receptor (A 3 AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A 3 AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A 3 AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A 3 AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A 3 AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([ 3 H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [ 3 H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H 1 receptor (H 1 R) antagonists were determined using radioligands with either slow (low k off ) or fast (high k off ) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

The influence of drug-receptor binding kinetics has often been overlooked during the development of new therapeutics that target G protein-coupled receptors (GPCRs). Over the last decade there has been a growing understanding that an in-depth knowledge of binding kinetics at GPCRs is required to successfully target this class of proteins. Ligand binding to a GPCR is often not a simple single step process with ligand freely diffusing in solution. This review will discuss the experiments and equations that are commonly used to measure binding kinetics and how factors such as allosteric regulation, rebinding and ligand interaction with the plasma membrane may influence these measurements. We will then consider the molecular characteristics of a ligand and if these can be linked to association and dissociation rates., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The human P2Y 2 receptor ( hP2Y 2 R) is a G-protein-coupled receptor that shows promise as a therapeutic target for many important conditions, including for antimetastatic cancer and more recently for idiopathic pulmonary fibrosis. As such, there is a need for new hP2Y 2 R antagonists and molecular probes to study this receptor. Herein, we report the development of a new series of non-nucleotide hP2Y 2 R antagonists, based on the known, non-nucleotide hP2Y 2 R antagonist AR-C118925 (1), leading to the discovery of a series of fluorescent ligands containing different linkers and fluorophores. One of these conjugates, 98, displayed micromolar affinity for hP2Y 2 R (p K d = 6.32 ± 0.10, n = 17) in a bioluminescence-energy-transfer (BRET) assay. Confocal microscopy with this ligand revealed displaceable membrane labeling of astrocytoma cells expressing untagged hP2Y 2 R. These properties make 98 one of the first tools for studying hP2Y 2 R distribution and organization.

Recent advances in the development of fluorescent ligands for G-protein-coupled receptors (GPCRs) and receptor tyrosine kinase receptors (RTKs) have facilitated the study of these receptors in living cells. A limitation of these ligands is potential uptake into cells and increased nonspecific binding. However, this can largely be overcome by using proximity approaches, such as bioluminescence resonance energy transfer (BRET), which localise the signal (within 10nm) to the specific receptor target. The recent engineering of NanoLuc has resulted in a luciferase variant that is smaller and significantly brighter (up to tenfold) than existing variants. Here, we review the use of BRET from N-terminal NanoLuc-tagged GPCRs or a RTK to a receptor-bound fluorescent ligand to provide quantitative pharmacology of ligand-receptor interactions in living cells in real time., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The histamine H 1 -receptor (H 1 R) is an important mediator of allergy and inflammation. H 1 R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H 1 R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H 1 R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H 1 R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications.

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A 3 AR. These were K114 (pK i 6.43), retinoic acid p -hydroxyanilide (pK i 6.13) and SU 6556 (pK i 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A 3 AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership., (Copyright © 2017. Published by Elsevier Ltd.)

A novel molecular scaffold has been synthesized, and its incorporation into new analogues of biologically active molecules across multiple target classes will be discussed. In these studies, we have shown use of the tricyclic scaffold to synthesize potent inhibitors of the serine peptidase DPP-4, antagonists of the CCR5 receptor, and highly potent and selective PI3K δ isoform inhibitors. We also describe the predicted physicochemical properties of the resulting inhibitors and conclude that the tractable molecular scaffold could have potential application in future drug discovery programs.

Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd ) and competition (Ki ) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super-bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Previous research has indicated that allosteric interactions across the dimer interface of β 1 -adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pK i values at the human β 1 -adenoceptor, which may result from such allosterism interactions. Three fluorescent β 1 -adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor-bound fluorescent ligand and the N-terminal NanoLuc tag of a human β 1 -adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high-affinity-specific binding to the NanoLuc- β 1 -adrenoceptor with each of the three fluorescent ligands yielding K D values of 87.1 ± 10 nmol/L ( n  = 8), 38.1 ± 12 nmol/L ( n  = 7), 13.4 ± 2 nmol/L ( n  = 14) for propranolol-Peg8-BY630, propranolol- β (Ala-Ala)-BY630 and CGP-12177-TMR, respectively. Parallel radioligand-binding studies with 3 H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc- β 1 -adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein -1 ). Following a 1 h incubation with fluorescent ligands and β 1 -adrenoceptor competing antagonists, there were significant differences ( P  < 0.001) in the pK i values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3 H-CGP 12177. However, increasing the incubation time to 2 h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand-receptor interactions at the human β 1 -adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made.

Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pKi values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology.

Fragment-based lead discovery (FBLD) holds great promise for drug discovery, but applications to G protein-coupled receptors (GPCRs) have been limited by a lack of sensitive screening techniques and scarce structural information. If virtual screening against homology models of GPCRs could be used to identify fragment ligands, FBLD could be extended to numerous important drug targets and contribute to efficient lead generation. Access to models of multiple receptors may further enable the discovery of fragments that bind specifically to the desired target. To investigate these questions, we used molecular docking to screen >500 000 fragments against homology models of the A3 and A1 adenosine receptors (ARs) with the goal to discover A3AR-selective ligands. Twenty-one fragments with predicted A3AR-specific binding were evaluated in live-cell fluorescence-based assays; of eight verified ligands, six displayed A3/A1 selectivity, and three of these had high affinities ranging from 0.1 to 1.3 μM. Subsequently, structure-guided fragment-to-lead optimization led to the identification of a >100-fold-selective antagonist with nanomolar affinity from commercial libraries. These results highlight that molecular docking screening can guide fragment-based discovery of selective ligands even if the structures of both the target and antitarget receptors are unknown. The same approach can be readily extended to a large number of pharmaceutically important targets.

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor-arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

G protein-coupled receptors control a wide range of physiological processes and are the target for many clinically used drugs. Understanding the way in which receptors bind agonists and antagonists, their organisation in the membrane and their regulation after agonist binding are important properties which are key to developing new drugs. One way to achieve this knowledge is through the use of fluorescent ligands, which have been used to study the expression and function of receptors in endogenously expressing systems. Fluorescent ligands with appropriate imaging properties can be used in conjunction with confocal microscopy to investigate the regulation of receptors after activation. Alternatively, through the use of single molecule microscopy, they can probe the spatial organisation of receptors within the membrane. This review focuses on the techniques in which fluorescent ligands have been used and the novel aspects of G protein-coupled receptor pharmacology which have been uncovered. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

Background and Purpose: The highly conserved tryptophan (W6.48) in transmembrane domain 6 of GPCRs has been shown to play a central role in forming an active conformation in response to agonist binding. We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A₃ receptor., Experimental Approach: Residue W6.48 in the human adenosine A₃ receptor fused to yellow fluorescent protein was mutated to phenylalanine and expressed in CHO-K1 cells containing a cAMP response element reporter gene. The effects on agonist-mediated receptor internalization were monitored by automated confocal microscopy and image analysis. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [(3)H]-cAMP accumulation and β-arrestin2 binding., Key Results: NECA was able to stimulate agonist-mediated internalization of the W6.48F mutant receptor, while the agonist HEMADO was inactive. Investigation of other downstream signalling pathways indicated that G-protein coupling was impaired for both agonists tested. Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor., Conclusions and Implications: Investigation of the pharmacology of the W6.48F mutant of the adenosine A₃ receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates., (Copyright © 2013 The British Pharmacological Society.)

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.

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-A(1) and -A(3) 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 (K(D)). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A(3) receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human dopamine D(2long) and D(3) receptors were modified by N-terminal addition of SNAP or CLIP forms of O(6)-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D(2long) and D(3) receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D(2long)-D(3) heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D(2long)-D(3) heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D(2long) and D(3) receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D(2long) and D(3) receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D(2long) and D(3) receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites.

The adenosine-A(3) receptor (A(3)AR) is a G protein-coupled receptor that shows promise as a therapeutic target for cancer, glaucoma, and various autoimmune inflammatory disorders, and as such, there is a need for molecular probes to study this receptor. Here, we report a series of fluorescent ligands containing different linkers and fluorophores based around a 1,2,4-triazolo[4,3-a]quinoxalin-1-one antagonist. One of these conjugates (19) displayed high affinity for the A(3)AR (pK(D) = 9.36 ± 0.12) and is >650-fold selective over other adenosine receptor subtypes. Confocal microscopy revealed clear, displaceable membrane labeling of CHO-A(3) cells with 19, with no detectable labeling of CHO-A(1) cells under identical conditions. This fluorescent ligand was also able to specifically label the A(3)AR in HEK293T cells containing a mixed adenosine receptor population. The subtype specificity, along with its excellent imaging properties, make 19 an ideal tool for studying A(3)AR distribution and organization, particularly in the presence of other adenosine receptor subtypes.

A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A(1) or A(3) adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A(3)-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A(1) and A(3) receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min(-1), respectively. At the A(3) receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5'-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A(3)-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A(1) receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC(50) values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A(3)-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu(173) or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Free fatty acid receptor 1 (FFA1; previously designated GPR40) is a potential therapeutic target for the treatment of diabetes and related metabolic disorders. Agonist-independent or constitutive activity is a feature associated with essentially all G protein-coupled receptors but the extent of this varies substantially between family members. In many situations, detection of such activity can be both assay- and context-dependent and may reflect the presence in the assay of an endogenous agonist. In studies on FFA1, experiments employing cell membrane preparations and the binding of [(35)S]guanosine 5'-O-[γ-thio]triphosphate to G proteins produce data consistent with a high-level constitutive activity of this receptor. Herein, we detail these assays and discuss approaches to determine if this is a measure of intrinsic receptor constitutive activity or if such results reflect the presence of an endogenous agonist. FFA1 is coupled predominantly to G proteins of the Gα(q) subfamily. Activation of the receptor results, therefore, in the transient elevation of intracellular [Ca(2+)]. We also detail assays to measure such signals and consider whether they are appropriate to detect receptor constitutive activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The free fatty acid receptors FFA2 and FFA3 are recently de-orphanized G protein-coupled receptors that share a group of short-chain free fatty acids as endogenous ligands. The expression of FFA2 and FFA3 by immune cells, in parts of the gastro-intestinal tract and by white adipocytes has suggested their potential as therapeutic targets in conditions including inflammation and obesity. However, although FFA2 and FFA3 display distinct structure-activity relationships for stimulation by short-chain free fatty acids, the overlap between these endogenous agonists and the lack of synthetic small molecule ligands that display selectivity between these two receptors has, until recently, hindered efforts to resolve their individual functions. Recently, chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide has been described as an FFA2 selective ago-allosteric ligand, not only being a direct agonist but also acting as a positive allosteric modulator of the function of short-chain free fatty acids at FFA2. Mutation of a pair of key arginine residues near the top of transmembrane domains V and VII of both FFA2 and FFA3 eliminates the function of short-chain free fatty acids but is without effect on the direct agonist action of chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide at FFA2, confirming the distinct nature of the binding site of the ago-allosteric regulator from the orthosteric binding site for free fatty acids. An understanding of structure-activity relationships for ligands related to chloro-alpha-(1-methylethyl)-N-2-thiazolylbenzeneacetamide is likely to provide greater insight into the mode of action and site of binding of this ligand, but further FFA2 and FFA3 selective ligands, preferably with higher potency/affinity, will be required to fully explore the physiological function of these receptors.

The endogenous ligands for free fatty acid receptor 1 (FFA1) are medium and longer chain free fatty acids. However, a range of selective, small molecule ligands have recently been developed as tool compounds to explore the therapeutic potential of this receptor, whereas clinically employed thiazolidinedione "glitazone" drugs are also agonists at FFA1. Each of these classes of agonist was able to promote phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases in cells able to express human FFA1 on demand. However, although both lauric acid and the synthetic agonist GW9508X produced rapid and transient ERK1/2 MAP kinase phosphorylation, the thiazolidinedione rosiglitazone produced responses that were sustained for a substantially longer period. Despite this difference, the effects of each ligand required FFA1 and were transduced in each case predominantly via G proteins of the Galphaq/Galpha11 family. Different glitazone drugs also displayed markedly different efficacy and kinetics of sustainability of ERK1/2 MAP kinase phosphorylation. A number of orthosteric binding site mutants of FFA1 were generated, and despite variations in the changes of potency and efficacy of the three ligand classes in different functional end point assays, these were consistent with rosiglitazone also binding at the orthosteric site. Four distinct polymorphic variants of human FFA1 have been described. Despite previous indications that these display differences in function and pharmacology, they all responded in entirely equivalent ways to lauric acid, rosiglitazone, and GW9508X in measures of ERK1/2 MAP kinase phosphorylation, enhancement of binding of [35S]GTPgammaS (guanosine 5'-O-(3-[35S]thio)triphosphate) to Galphaq, and elevation of intracellular [Ca2+], suggesting that individuals expressing each variant are likely to respond equivalently to orthosteric agonists of FFA1.

Identification of G protein-coupled receptors that are activated by free fatty acids has led to considerable interest in their pharmacology and function because of the wide range of normal physiology and disease states in which fatty acids have been implicated. Free fatty acid receptor (FFA) 1 is activated by medium- to long-chain fatty acids and is expressed in the insulin-producing beta-cells of the pancreas. Activation of FFA1 has been proposed to mediate fatty acid augmentation of glucose-stimulated insulin secretion although it is unclear whether the known long-term detrimental effects of beta-cell exposure to high levels of fatty acids are also mediated through this receptor. The related receptors FFA2 and FFA3 are both activated by short-chain fatty acids although they have key differences in the signaling pathways they activate and tissue expression pattern. The aim of this review is to provide a comprehensive overview of the current understanding of the pharmacology and physiological role of these fatty acid receptors.

FFA2 and FFA3 are closely related G protein-coupled receptors that bind and respond to short chain fatty acids. (FFA2 and FFA3 are the provisional International Union of Pharmacology designations for the receptors previously called GPR43 and GPR41, respectively.) Sequence comparisons between these two receptors and alignments with the related G protein-coupled receptor FFA1, linked to homology modeling based on the atomic level structure of bovine rhodopsin, indicated the potential for polar residues within the transmembrane helix bundle to play important roles in ligand recognition and function. In both FFA2 and FFA3, mutation of either an arginine at the top of transmembrane domain V or a second arginine at the top of transmembrane domain VII eliminated the function of a range of short chain fatty acids. Mutation of a histidine in transmembrane domain VI, predicted to be in proximity to both the arginine residues, also eliminated function in many but not all assay formats. By contrast, mutation of a histidine in transmembrane domain IV, predicted to be lower in the binding pocket, modulated function in some assays of FFA3 function but had limited effects on the function of acetate and propionate at FFA2. Interestingly, wild type FFA3 responded to caproate, whereas FFA2 did not. Mutation of the transmembrane domain IV histidine eliminated responses of FFA3 to caproate but resulted in a gain of function of FFA2 to this six-carbon fatty acid. These data demonstrate the importance of positively charged residues in the recognition and/or function of short chain fatty acids in both FFA2 and FFA3. The development of small molecule ligands that interact selectively with these receptors will allow further details of the binding pockets to be elucidated.

Single, bi-functional polypeptides consisting of a G-protein-coupled receptor (GPCR) linked directly to a G protein alpha subunit have been employed for a number of years to study many aspects of signal initiation, including the roles of post-translational modifications, effects of mutations in both receptor and G protein and in the de-orphanisation of novel G-protein-coupled receptors. Recently, they have been used to improve signal-to-background in ligand assay screens and to study both agonist-directed signal trafficking and distinct conformational states of receptors. As well as such novel concepts in pharmacology, G-protein-coupled receptor-G protein fusions have recently been employed to examine receptor homo-dimerisation and hetero-dimerisation and are beginning to be used to explore allosteric effects within GPCR hetero-dimers.

In cells lacking expression of Ca(2+)-mobilizing G proteins, coexpression of human GPR40 and Galpha(q) allowed medium- and long-chain fatty acids to elevate intracellular [Ca(2+)]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Galpha(q) fusion protein. The kinetic of elevation of intracellular [Ca(2+)] slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whereas the addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effective ligand equilibration, GPR40-Galpha(q) was used in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays. After expression of GPR40-Galpha(q) in HEK293 cells and membrane preparation basal binding of [(35)S]GTPgammaSinGalpha(q) immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [(35)S]GTPgammaS binding in a concentration-dependent manner and allowed the responsiveness and pharmacology at GPR40 of each of the fatty acids thiazolidinediones and a novel small-molecule agonist to be uncovered. Membranes of rat INS-1E cells that express GPR40 endogenously provided similar observations. The high apparent constitutive activity of GPR40-Galpha(q) was also reversed by a small-molecule GPR40 antagonist, and basal [(35)S]GTPgammaS binding was prevented by the selective Galpha(q)/Galpha(11) inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors which respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists before assay and that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.

Free fatty acids (FFAs) are not only an important direct source of energy but they also play key roles in regulating a range of physiological responses. Although it was long assumed that such effects of FFAs must occur following cellular uptake, and potentially via their conversion to fatty acyl-CoAs, it is now apparent that FFAs also function directly as agonists at a number of G protein-coupled receptors (GPCRs). Tissue distribution studies and, subsequently, siRNA knock-down experiments have indicated key roles for these GPCRs in glucose homeostasis, adipogenesis, white cell recruitment and potentially in a range of other processes. Considerable interest is thus now centred on the generation of potent and selective small molecule ligands, both as tool compounds to further unravel the biology and physiological role of this group of GPCRs and as starting points for possible therapeutic intervention in a range of areas, particularly those associated with 'metabolic syndrome'.