Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures (original) (raw)
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Ligand-selective receptor conformations revisited: the promise and the problem
Trends in Pharmacological Sciences, 2003
Ligand-selective receptor conformations introduce the concept of 'texture' to drug effects, with respect to ligands possessing quality in addition to quantity of efficacy. This cell-dependent phenotypic efficacy extends to ligand properties beyond G-protein signaling and, in terms of drug development, presents a twoedged sword to pharmacologists. On the one hand, such efficacy promises more selective agonism but on the other hand it predicts problems associated with the use of recombinant or natural lead optimization assays as predictors of therapeutic value in humans. In this article, the evidence to suggest that not all agonists produce the same receptor active state is reviewed.
Journal of Medicinal Chemistry, 2009
Homology modeling of the human A 2A adenosine receptor (AR) based on bovine rhodopsin predicted a protein structure that was very similar to the recently determined crystallographic structure. The inaccuracy of previous antagonist docking is related to the loop structure of rhodopsin being carried over to the model of the A 2A AR and was rectified when the β 2-adrenergic receptor was used as a template is used for homology modeling. Docking of the triazolotriazine antagonist ligand ZM241385 1 was greatly improved by including water molecules of the X-ray structure or by using a constraint from mutagenesis. Automatic agonists docking to both a new homology modeled receptor and the A 2A AR crystallographic structure produced similar results. Heterocyclic nitrogen atoms closely corresponded when the docked adenine moiety of agonists and 1 were overlayed. The cumulative mutagenesis data, which support the proposed mode of agonist docking, can be reexamined in light of the crystallographic structure. Thus, homology modeling of GPCRs remains a useful technique in probing the structure of the protein and predicting modes of ligand docking.
The impact of GPCR structures on pharmacology and structure-based drug design
British Journal of Pharmacology, 2010
After many years of effort, recent technical breakthroughs have enabled the X-ray crystal structures of three G-protein-coupled receptors (GPCRs) (b1 and b2 adrenergic and adenosine A2a) to be solved in addition to rhodopsin. GPCRs, like other membrane proteins, have lagged behind soluble drug targets such as kinases and proteases in the number of structures available and the level of understanding of these targets and their interaction with drugs. The availability of increasing numbers of structures of GPCRs is set to greatly increase our understanding of some of the key issues in GPCR biology. In particular, what constitutes the different receptor conformations that are involved in signalling and the molecular changes which occur upon receptor activation. How future GPCR structures might alter our views on areas such as agonist-directed signalling and allosteric regulation as well as dimerization is discussed. Knowledge of crystal structures in complex with small molecules will enable techniques in drug discovery and design, which have previously only been applied to soluble targets, to now be used for GPCR targets. These methods include structure-based drug design, virtual screening and fragment screening. This review considers how these methods have been used to address problems in drug discovery for kinase and protease targets and therefore how such methods are likely to impact GPCR drug discovery in the future. 159 986-996
Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation
Nature, 2011
The adenosine receptors and β-adrenoceptors (βARs) are G protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding agonist such as adenosine 1 or noradrenaline 2 , respectively. GPCRs have similar structures consisting of 7 transmembrane helices that contain well-conserved sequence motifs, suggesting that they are probably activated by a common mechanism 3,4 . Recent structures of βARs highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, suggesting an important role in agonist-induced activation of receptors 5-7 . Here we present two crystal structures of the thermostabilised human adenosine A 2A receptor (A 2A R-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 bind in a similar fashion to the chemically-related region of the inverse agonist ZM241385 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 (Ser277 7.42 and His278 7.43 ; superscripts refer to Ballesteros-Weinstein numbering 9 ) 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 suggests that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A 2A R with the agonist-bound structures of β-adrenoceptors suggests 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.
2009
G protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes, ranging from the transmission of the light and odorant signals to the mediation of neurotransmission and hormonal actions. GPCRs are dysfunctional or deregulated in several human diseases and are estimated to be the target of more than 40% of drugs used in clinical medicine today. The crystal structures of rhodopsin and the recent published crystal structures of beta-adrenergic receptors and human A2A Adrenergic Receptor provide the information of the three-dimensional structure of GPCRs, which supports homology modeling studies and structure-based drug-design approaches. Rhodopsin-based homology modeling has represented for many years a widely used approach to built GPCR three-dimensional models. Structural models can be used to describe the interatomic interactions between ligand and receptor and how the binding info...
Journal of The American Chemical Society, 2010
G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the 2 adrenergic receptor ( 2 AR) is one of the most extensively studied. Previously, the X-ray crystal structure of 2 AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered 2 AR construct in complex with two inverse agonists: ICI 118,551 (2.8 Å), a recently described compound (2.8 Å) , and the antagonist alprenolol (3.1 Å). The structures show the same overall fold observed for the previous 2 AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with 2 AR. Furthermore, receptor ligand cross-docking experiments revealed that a single 2 AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.
2019
Adenosine A 3 receptor (A 3 R), which is activated by adenosine (Ado, (1)), is over-expressed in various tumor cells and it is a promising drug target against cancer cell proliferation and other conditions including asthma, rheumatoid arthritis and ischemic injury. Currently there is no experimental structure of A 3 R and in this work the orthosteric binding site of A 3 R in complex with two agonists, the non-selective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-Dribofuranuronamide (NECA, (2)) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA, (3)) was studied. Molecular dynamics simulations (MD) of the wild-type (WT) A 3 R in complex with NECA (2) or IB-MECA (3) were performed to identify the residues important for binding in the orthosteric site and several mutagenic studies were conducted to investigate the agonists binding profile. The Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) free energy calculations were able to distinguish mutations that reduce or negate NECA (2) or IB-MECA (3) activity from those that maintained or increased activity. The calculated ΔG eff values for both IB-MECA and NECA (2) displayed good correlations with experimental activities. The combined computational and experimental results suggested that agonist binding and receptor activation is realized through direct interactions with residues of the orthosteric area, such as π-π interactions with F168 5.29 , van der Waals interactions with L246 6.51 and I268 7.39 , and hydrogen bond interactions with T94 3.36 , N250 6.55 S271 7.42 and H272 7.43. Mutating these residues to alanine negated agonist activity. Alanine mutation of the directly interacting residues W185 5.46 , L264 7.35 maintained activity for both agonists although mutation of V169 5.30 increased NECA (2) activity but maintained IB-MECA (3). The selectivity of IB-MECA (3) against A 3 R is not only due to direct interactions with the binding area residues, but also due to indirect effects, through residues positioned at the extracellular loop 2, transmembrane domains 5 and 6, as well as deeper in the orthosteric binding area. Indirect interactions including residues L90 3.32 , M177 5.38 were critical for both agonists binding, M174 5.35 was important only for NECA (2), and I253 6.58 was unimportant for agonists binding. Moreover, although V169 5.30 is considered to be a selectivity filter for A 3 R binders, when this residue was mutated to glutamic acid, the activity of IB-MECA (3) against A 3 R increased. The study aimed at highlighting features of the still-unsolved A 3 R that are important for IB-MECA (3)and NECA (2) binding and may be used for the design of effective ligands.
Journal of Computer-Aided Molecular Design, 2006
It is well known that G protein-coupled receptors are prime targets for drug discovery. At the present time there is only one protein from this class that has an X-ray crystal structure, bovine rhodopsin. Crystal structures of rhodopsin have become invaluable templates for the modeling of class-A G proteincoupled receptors as they likely represent the overall topology of this family of proteins. However, because of low sequence homology within the class and the inherent mobility of integral membrane proteins, it is unlikely that this single structural template reflects the ensemble of conformations accessible for any given receptor. We have devised a procedure based upon comparative modeling that uses induced fit modeling coupled with binding site expansion. The modeling protocol enables an ensemble approach to binding mode prediction. The utility of models for b-2 adrenergic receptor will be discussed.