Jillian G. Baker

Structure of a Beta1-Adrenergic G-Protein-Coupled Receptor.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

The structural basis for agonist and partial agonist action on a β1-adrenergic receptor

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46), but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros–Weinstein numbering). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies.

The selectivity of β‐adrenoceptor antagonists at the human β1, β2 and β3 adrenoceptors

1 β‐Adrenoceptor antagonists (‘β‐blockers’) are one of the most widely used classes of drugs in cardiovascular medicine (hypertension, ischaemic heart disease and increasingly in heart failure) as well as in the management of anxiety, migraine and glaucoma. Where known, the mode of action in cardiovascular disease is from antagonism of endogenous catecholamine responses in the heart (mainly at β1‐adrenoceptors), while the worrisome side effects of bronchospasm result from airway β2‐adrenoceptor blockade. The aim of this study was to determine the selectivity of β‐antagonists for the human β‐adrenoceptor subtypes. 2 3H‐CGP 12177 whole cell‐binding studies were undertaken in CHO cell lines stably expressing either the human β1‐, β2‐ or the β3‐adrenoceptor in order to determine the affinity of ligands for each receptor subtype in the same cell background. 3 In this study, the selectivity of well‐known subtype‐selective ligands was clearly demonstrated: thus, the selective β1 antagonist CGP 20712A was 501‐fold selective over β2 and 4169‐fold selective over β3; the β2‐selective antagonist ICI 118551 was 550‐ and 661‐fold selective over β1 and β3, respectively, and the selective β3 compound CL 316243 was 10‐fold selective over β2 and more than 129‐fold selective over β1. 4 Those β2‐adrenoceptor agonists used clinically for the treatment of asthma and COPD were β2 selective: 29‐, 61‐ and 2818‐fold for salbutamol, terbutaline and salmeterol over β1, respectively. There was little difference in the affinity of these ligands between β1 and β3 adrenoceptors. 5 The clinically used β‐antagonists studied ranged from bisoprolol (14‐fold β1‐selective) to timolol (26‐fold β2‐selective). However, the majority showed little selectivity for the β1‐ over the β2‐adrenoceptor, with many actually being more β2‐selective. 6 This study shows that the β1/β2 selectivity of most clinically used β‐blockers is poor in intact cells, and that some compounds that are traditionally classed as ‘β1‐selective’ actually have higher affinity for the β2‐adrenoceptor. There is therefore considerable potential for developing more selective β‐antagonists for clinical use and thereby reducing the side‐effect profile of β‐blockers.

The selectivity of beta-adrenoceptor agonists at human beta1-, beta2- and beta3-adrenoceptors.

Background and purpose:  There are two important properties of receptor–ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor–ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 β‐adrenoceptor agonists at the three human β‐adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.

Structure of a β1-adrenergic G protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Structure of a |[bgr]|1-adrenergic G-protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Reporter-gene systems for the study of G-protein-coupled receptors

Reporter-gene assays offer an alternative to biochemical assays for following signal transduction pathways from receptors at the cell surface to nuclear gene transcription in living cells. Specific reporter-gene systems are now available for the study of ligand activity at G alpha(i/o), G alpha(s) and G alpha(q) G-protein-coupled receptors. In recent years reporter genes have been applied in academia and industry to the study of ligand efficacy and affinity in recombinant and primary cell lines using a variety of colour, fluorescent or luminescent read-outs.

Evolution of β-blockers: from anti-anginal drugs to ligand-directed signalling

Sir James Black developed β-blockers, one of the most useful groups of drugs in use today. Not only are they being used for their original purpose to treat angina and cardiac arrhythmias, but they are also effective therapeutics for hypertension, cardiac failure, glaucoma, migraine and anxiety. Recent studies suggest that they might also prove useful in diseases as diverse as osteoporosis, cancer and malaria. They have also provided some of the most useful tools for pharmacological research that have underpinned the development of concepts such as receptor subtype selectivity, agonism and inverse agonism, and ligand-directed signalling bias. This article examines how β-blockers have evolved and indicates how they might be used in the future.

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.

Pharmacological characterization of CGP 12177 at the human β2-adrenoceptor

It has recently been reported that CGP 12177 can act as an agonist at a novel secondary site within the human β1‐adrenoceptor. The aim of this study was to undertake a detailed pharmacological study of the effects of CGP 12177 on the human β2‐adrenoceptor. CGP 12177 acted as a potent partial agonist of 3H‐cyclic AMP accumulation (log EC50−8.90±0.06) and CRE‐mediated reporter gene transcription (log EC50−9.66±0.04) in CHO‐K1 cells expressing the human β2‐adrenoceptor. These CGP‐induced responses were antagonized by the β2‐selective antagonist ICI 118551 (apparent log KD values of −8.84±0.15 and −9.51±0.02 for the cyclic AMP and reporter gene responses respectively). CGP 12177 was also able to antagonize both cyclic AMP and reporter gene responses to more efficacious β2‐agonists with similar log KD values (e.g. −9.57±0.15 and −10.04±0.096 respectively with salbutamol as agonist). 3H‐CGP 12177 binding to β2‐adrenoceptors in intact CHO‐β2 cells yielded a log KD value of −9.84±0.06, but indicated that the ligand dissociates very slowly from the receptor (t½ for dissociation=65 min). However, studies with a Green Fluorescent Protein (GFP)‐tagged β2‐adrenoceptor indicated that CGP 12177 does not stimulate β2‐adrenoceptor internalization. This study demonstrates that CGP 12177 is a high affinity partial agonist of both cAMP accumulation and CRE‐mediated gene transcription at the human β2‐adrenoceptor. It provides no evidence that CGP 12177 can discriminate a secondary site on the β2‐adrenoceptor analogous to that observed for the human β1‐adrenoceptor. However, despite its very weak actions on cAMP accumulation, the potent agonist effects of CGP 12177 on CRE‐mediated gene transcription at the human β2‐adrenoceptor, coupled with its long duration of action, offers a potential lead for drug development for the treatment of chronic inflammatory airway diseases.