Docking studies of agonists and antagonists suggest an activation pathway of the A3 adenosine receptor
Introduction
Four subtypes of adenosine receptors (ARs), a rhodopsin-like Family A G-protein-coupled receptor (GPCR), have been important therapeutic targets for drug development [1]. Agonists of the A3AR subtype act to prevent ischemic damage in the brain and heart, and have anti-inflammatory, anti-cancer and myeloprotective effects. The compound IB-MECA (N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine), which we developed as a selective A3AR agonist, has been in Phase II clinical studies for the treatment of metastatic colorectal tumors and rheumatoid arthritis [2], [3].
However, there are major barriers to the clinical development of AR agonists. The ubiquitous expression of ARs in the body leads to diverse side effects, and the low receptor density in the targeted tissue limits the effect of drugs in certain diseases. A3AR selective agonists have been described as promising cardio- and cerebroprotective agents. However, the low density of the A3AR in the heart may be a problem [4]. In 2000, it was reported that one of the major causes of attrition in drug discovery, accounting for 28% of cases, was the lack of systemic drug efficacy [5]. Thus, to develop selective A3AR agonists with the aid of molecular modeling, the structural requirements for subtype selectivity and the molecular properties required for agonism should be studied. Understanding the molecular mechanism involved in agonist binding and the accompanying conformational change of the A3ARs is also crucial.
The 3D-structures of GPCRs provide important information for understanding their molecular organization and how agonists participate in conformational changes upon activation. However, obtaining the structural information about GPCRs through the application of standard structural determination techniques, X-ray, and NMR studies, has progressed slowly. The main reason has been the technical difficulty of large-scale receptor purification and the insolubility in media lacking phospholipids. However, since the X-ray structure of bovine rhodopsin (PDB ID: 1F88) was first published in 2000 [6], this structure has been widely applied to homology modeling, based on the hypothesis of structural mimicry, i.e. different amino acids or alternate microdomains can support similar deviations from a regular α-helical structure, thereby resulting in a similar tertiary structure [7].
To study the activation mechanism of GPCRs, several limitations of computer modeling as well as experimental approaches have been considered: (1) Currently, the unique template for generating 3D-models of GPCRs is the inactive form of bovine rhodopsin to which cis-retinal is covalently bound as an inverse agonist. The reliability of docking models of agonists is limited by using a ground-state template. (2) Although several computational models of the active state [8], [9] have been proposed, these theoretical models all exhibited different conformations depending on the experimental data. Some models were not consistent with recent experimental results, e.g. the computational model did not show the predicted separation of the cytosolic extensions of TMs 3 and 6 [10]. (3) Explicit membrane/water added to a model makes it more difficult to perform molecular dynamics (MD) calculations with millisecond time scales in order to simulate the active states. (4) It is difficult to study drug efficacy with only a single snapshot from the several theoretically generated active conformations [8], [9], [10]. Receptors likely exist as collected ensembles of numerous conformations. Fluorescence spectroscopy studies of the β2-adrenergic receptor provided evidence for a multi-step process of agonist binding, identifying the order of contacts between the receptor and key moieties [11]. A single agonist stabilizes a succession of conformational states with distinct cellular functions through induction or stabilization of multiple, functionally distinct conformational states. (5) The experimentally-determined relative efficacy of a given agonist may reflect interactions within higher order GPCR networks, a communication system through various interactions with the surroundings. The calculation may require considering the complexes of GPCR oligomers and G-proteins. Thus, a single active receptor model would not likely lead to an understanding of the efficacy of various ligands and the activation mechanism of a given GPCR.
However, the structure-activity relationship (SAR) of a wide range of adenosine derivatives at the A3AR suggests the possibility of rationally “tuning” a desired selectivity and activity through structural modification [12], [13]. Activation of the A3AR depends on structural determinants of relative efficacy, independent of binding affinity, in adenosine derivatives. In this study, as depicted in Fig. 1, we compared antagonists and agonists according to their molecular properties and by phamacophore analysis. Complementary to ligand SAR, extensive mutagenesis studies, interpreted through AR homology modeling, have identified amino acids involved in ligand binding and activation [14]. In this study we have used the structural, pharmacological, and physical/chemical properties of these ligands to discern likely steps in the activation process.
We explored the different binding domain preferences and compared the specific interactions of representative A3 selective ligands having three distinct functions, e.g. an inverse agonist, a neutral antagonist, and a full agonist. The difference in the preference of binding domain and/or local conformational change depended on the type of ligand docked. Representative antagonists were docked to an A3AR conformation based on the X-ray structure of the resting state of rhodopsin, and agonists were docked to a putative Meta I state conformation of the A3AR (Fig. 1). Thus, the A3AR docking study of different categories of ligands provided a hypothetical mechanism of the initial conformational step(s) in receptor activation.
The agonist-bound conformation, in a form resembling the not fully-activated Meta I state of rhodopsin, was obtained by modeling the rearrangement of the side-chain of a key conserved Trp residue of ARs in transmembrane (TM) helical domain 6 (6.48). This rearrangement was required in order to dock various A3AR agonists and was not required for antagonist docking. Although the Meta I state is still far more similar to the resting conformation than to the presumed, yet undisclosed fully active conformation, the Meta I state structure is preferable to the ground-state structure for agonist docking. The agonist-bound state of the A3AR is similar to the Meta I state of rhodopsin, which is not the result of large rigid-body movements of helices, but rather a rearrangement of side-chains, especially 6.48, through a local conformational change in the binding site [15]. Consequent to the rearrangement of side-chains upon activation of GPCRs is the disruption of intramolecular interactions that constrain the receptor in the inactive state. Agonist binding to the A3AR appears to disrupt the intramolecular H-bonding networks involving W6.48 and H7.43 and the specific interactions at highly conserved T3.36, S7.42, and H7.43, to induce a characteristic anti-clockwise movement of TMs 3, 6, and 7 from the extracellular view. Thus, we present novel insights into a putative activation mechanism of the A3AR based on correlation of ligand recognition patterns in receptor docking and pharmacological function.
Section snippets
Summary of relevant reported SAR of A3AR antagonists and agonists
A number of adenosine derivatives have been developed as A3AR selective agonists. Binding to the human A3AR (hA3AR) was characterized pharmacologically using a high affinity radioligand, [125I]I-AB-MECA, and receptor activation was measured as the inhibition of forskolin-stimulated adenylate cyclase in intact CHO (Chinese hamster ovary) cells stably expressing this receptor [16].
Discussion
All GPCRs have common structural components, including seven TM-spanning α-helical segments connected by alternating intracellular and extracellular loops (ELs), with the amino terminus on the extracellular side and the carboxyl terminus on the intracellular side. Although the overall sequence homology among all Family A receptors is less than 20%, sequence analysis suggested that Family A GPCRs could share the same arrangement of the seven helices in the plane of lipid bilayers, because of the
Conclusions
The molecular modeling results clearly delineated the interactions involved in the binding of agonists and antagonists, which correlated well with known experimental results. The calculation of molecular properties and the study of pharmacophores indicated the characteristic differences between agonists and antagonists. The docking complexes provided insight into the conformational and binding requirements for agonists and antagonists at the A3AR. Combination of the docking studies and
Molecular modeling
All calculations were performed on a Silicon Graphics (Mountain View, CA) Octane workstation (300 MHz MIPS R12000 (IP30) processor). All ligand structures were constructed with the use of the Sketch Molecule of SYBYL 7.0 [43].
The 3D-structure and pharmacophore analysis of ligands
A conformational search of each ligand was performed by random search of flexible bonds. The low-energy conformers from the random search were re-optimized, removing all constraints. The options of random search for all rotatable bonds were 3000 iterations, 3-kcal energy
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases. We thank Dr. Jürgen Wess (NIDDK) for helpful discussions.
References (53)
- et al.
A3 adenosine receptor activation in melanoma cells: association between receptor fate and tumor growth inhibition
J. Biol. Chem.
(2003) - et al.
Receptor activation: what does the rhodopsin structure tell us?
Trends Pharmacol. Sci.
(2001) - et al.
Sequential binding of agonists to the β2 adrenoceptor
J. Biol. Chem.
(2004) - et al.
N6-Substituted adenosine derivatives: selectivity, efficacy, and species differences at A3 adenosine receptor
Biochem. Pharmacol.
(2003) - et al.
Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position
Bioorg. Med. Chem.
(2004) - et al.
Exploring distal regions of the A3 adenosine receptor binding site: sterically constrained N6-(2-phenylethyl)adenosine derivatives as potent ligands
Bioorg. Med. Chem.
(2004) - et al.
Conversion of A3 adenosine receptor agonists into selective antagonists by modification of the 5′-ribofuran-uronamide moiety
Bioorg. Med. Chem. Lett.
(2006) - et al.
Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety
Biochem. Pharmacol.
(2004) - et al.
Structure-activity relationships of thiazole and thiadiazole derivatives as potent and selective human adenosine A3 receptor antagonists
Bioorg. Med. Chem.
(2004) - et al.
2-Phenylimidazo[2,1-i]purin-5-ones: structure-activity relationships and characterization of potent and selective inverse agonists at human A3 adenosine receptors
Bioorg. Med. Chem.
(2003)
Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes
J. Biol. Chem.
Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy
J. Biol. Chem.
Identification of an agonist-induced conformational change occurring adjacent to the ligand binding pocket of the M3 muscarinic acetylcholine receptor
J. Biol. Chem.
Structure of bovine rhodopsin in a trigonal crystal form
J. Mol. Biol.
Pronounced conformational changes following agonist activation of the M3 muscarinic acetylcholine receptor
J. Biol. Chem.
The exclusive nature of intrinsic efficacy
Trends Pharmacol. Sci.
Binding thermodynamics at the human A3 adenosine receptor
Biochem. Pharmacol.
A fast flexible docking method using an incremental construction algorithm
J. Mol. Biol.
Design, synthesis and binding affinity of 3′-fluoro analogues of Cl-IB-MECA as adenosine A3 receptor ligands
Bioorg. Med. Chem. Lett.
Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cell
Biochem. Pharmacol.
Adenosine receptors as therapeutic targets
Nature Rev. Drug Disc.
Purine derivatives as ligands for A3 adenosine receptors
Curr. Top Med. Chem.
Adenosine receptor agonist: from basic medicinal chemistry to clinical development
Expert Opin. Emerg. Drugs
Crystal structure of rhodopsin: a G protein-coupled receptor
Science
Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors
Mol. Pharmacol.
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