Christopher J. Langmead

Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation

Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism. Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors. Here we present two crystal structures of the thermostabilized human adenosine A2A receptor (A2AR-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 2777.42 and His 2787.43; superscripts refer to Ballesteros–Weinstein numbering) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A2AR with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

Discovery of 1,2,4-Triazine Derivatives as Adenosine A(2A) Antagonists using Structure Based Drug Design

Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A2A receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson’s disease.

Progress in Structure Based Drug Design for G Protein-Coupled Receptors

In 1998, Bikker, Trumpp-Kallmeyer, and Humblet published a Perspective in this journal entitled “G-Protein Coupled Receptors: Models, Mutagenesis and Drug Design” and reviewed the state of the art at that time.1 No high resolution structure of a G protein-coupled receptor (GPCR) had been solved, and researchers were working with models generated with only the structure of bacteriorhodopsin,2 which had been published 8 years earlier and solved using high resolution electron cryomicroscopy and the low resolution electron density footprint of bovine rhodopsin.3 These models, despite greatly improving understanding of GPCR structure and function, posed as many questions as they answered and were not able to clearly rationalize how ligands bound to their target receptor. The authors stated “The principal limitation of the current generation of models when used for rational drug design is that the resolution of the binding cavity is too low to predict specific ligand–receptor interactions. Attempts to dock ligands into various GPCR models are further complicated by difficulty in identifying unique, sensible modes of binding, especially when dealing with molecules of the size of the neurotransmitter ligands.” How things have changed. Today, there are six GPCRs for which medium to high resolution crystal structures have been solved, in most cases with multiple small molecules ligands. The six receptors are rhodopsin, the β1 and β2 adrenergic receptors, adenosine A2A receptor, chemokine CXCR4 receptor, and dopamine D3 receptor (Table ​(Table11 and references therein). In addition, rhodopsin, the β1 and β2 adrenergic receptors (ARs), and the adenosine A2A receptor have been solved with both antagonists and agonists bound (Table ​(Table1).1). Much current research is now engaged in using this new body of structural information for hit identification and drug design purposes, and we will review the state of the art of both structures and the impact they are now having on structure based drug design (SBDD) for GPCR targets in this article. Table 1 List of Published GPCR Crystal Structures

Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor

This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB‐674042 (1‐(5‐(2‐fluoro‐phenyl)‐2‐methyl‐thiazol‐4‐yl)‐1‐((S)‐2‐(5‐phenyl‐(1,3,4)oxadiazol‐2‐ylmethyl)‐pyrrolidin‐1‐yl)‐methanone), to the human orexin‐1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane‐based scintillation proximity assay (SPA) format. Specific binding of [3H]SB‐674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high‐affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein−1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin‐A displaying a ∼five‐fold higher affinity than orexin‐B (Ki values of 318±158 and 1516±597 nM, respectively). SB‐334867, SB‐408124 (1‐(6,8‐difluoro‐2‐methyl‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) and SB‐410220 (1‐(5,8‐difluoro‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB‐334867, SB‐408124 and SB‐410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50‐fold selectivity over the orexin‐2 receptor. These studies indicate that [3H]SB‐674042 is a specific, high‐affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors.

The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery.

G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A(2A) receptor and muscarinic M(1) receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A(2A)-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein–coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172EDGE175, Tyr177, and Thr423. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr177 and Thr423, rather than the 172EDGE175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1

M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimers disease and schizophrenia. However, the discovery of subtype‐selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh‐binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl]‐piperidine), which bind to an allosteric site and selectively activate the M1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs.

Identification of novel adenosine A(2A) receptor antagonists by virtual screening.

Virtual screening was performed against experimentally enabled homology models of the adenosine A2A receptor, identifying a diverse range of ligand efficient antagonists (hit rate 9%). By use of ligand docking and Biophysical Mapping (BPM), hits 1 and 5 were optimized to potent and selective lead molecules (11–13 from 5, pKI = 7.5–8.5, 13- to >100-fold selective versus adenosine A1; 14–16 from 1, pKI = 7.9–9.0, 19- to 59-fold selective).

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1 Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M1 receptor previously suggested to interact with an “ectopic” site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M1 receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [3H]N-methylscopolamine ([3H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [3H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [3H]NMS dissociation from CHO-hM1 cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M1 receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M1 receptors in the absence of the orthosteric agonist.