Rat A3 Adenosine Receptor Research Articles

Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2

Besides classical G protein coupling, G protein-coupled receptors (GPCRs) are nowadays well known to show significant signalling via other adaptor proteins, such as β-arrestin2 (βarr2). The elucidation of the molecular mechanism of the GPCR-βarr2 interaction is a prerequisite for the structure-activity based design of biased ligands, which introduces a new chapter in drug discovery. The general mechanism of the interaction is believed to rely on phosphorylation sites, exposed upon agonist binding. However, it is not known whether this mechanism is universal throughout the GPCR family or if GPCR-specific patterns are involved. In recent years, promising orally active agonists for the human A3 adenosine receptor (A3AR), a GPCR highly expressed in inflammatory and cancer cells, have been evaluated in clinical trials for the treatment of rheumatoid arthritis, psoriasis, and hepatocellular carcinoma. In this study, the effect of cytoplasmic modifications of the A3AR on βarr2 recruitment was evaluated in transiently transfected HEK293T cells, using a live-cell split-reporter system (NanoBit®, Promega), based on the structural complementation of NanoLuc luciferase, allowing real-time βarr2 monitoring. The A3AR-selective reference agonist 2-Cl-IB-MECA yielded a robust, concentration dependent (5 nM–1 µM) recruitment of βarr2 (logEC50: −7.798 ± 0.076). The role of putative phosphorylation sites, located in the C-terminal part and cytoplasmic loops, and the role of the ‘DRY’ motif was evaluated. It was shown that the A3AR C-terminus was dispensable for βarr2 recruitment. This contrasts with studies in the past for the rat A3AR, which pointed at crucial C-terminal phosphorylation sites. When combining truncation of the A3AR with modification of the ‘DRY’ motif to ‘AAY’, the βarr2 recruitment was drastically reduced. Recruitment could be partly rescued by back-mutation to ‘NQY’, or by extending the C-terminus again. In conclusion, other parts of the human A3AR, either cytosolic or exposed upon receptor activation, rather than the C-terminus alone, are responsible for βarr2 recruitment in a complementary or synergistic way.

Synthesis and pharmacological characterization of [125I]MRS1898, a high-affinity, selective radioligand for the rat A3 adenosine receptor

A known selective agonist of the A3 adenosine receptors (AR), MRS1898 [(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A3AR affinity across species, with nanomolar binding at both rat and human A3ARs. The radioiodination of MRS1898 on its N6–3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [125I]MRS1898 bound to the rat A3AR with a Kd value of 0.17 ± 0.04 nM and a Bmax value of 0.66 ± 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [125I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A3AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A3AR.

Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2, 4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists.

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists at both human and rat A3 adenosine receptors (Li et al. J. Med. Chem. 1998, 41, 3186-3201). In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values at recombinant human and rat A3 adenosine receptors were determined using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine). Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors, and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a beta-fluoroethyl (7) or a gamma-fluoropropyl ester (26) was favorable for human A3 receptor affinity, resulting in Ki values of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue was clearly less potent at human A3 receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy substitution failed to enhance potency and selectivity at human A3 receptors. Several analogues were nearly equipotent at rat and human A3 receptors. To further define the pharmacophore conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor binding. The most potent analogue in this group was compound 34, in which a thiolactone was formed between 3- and 4-positions and which had a Ki value of 248 nM at human A3 receptors. Using affinity data and a general pharmacophore model for A3 adenosine receptor antagonists recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-dimensional quantitative structure-activity relationship for pyridine derivatives, having good predictability (r2pred = 0.873) for compounds in the test set. A rhodopsin-based model of the human A3 receptor was built, and the pyridine reference ligand 2,3,4, 5-tetraethyl-6-phenyl-pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding site. Interactions between receptor transmembrane domains and the steric and the electrostatic contour plots obtained from the CoMFA analysis were analyzed.

Synthesis and biological activity of a new series of N6-arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-5'-N-ethyluronamide as A1 and A3 adenosine receptor agonists.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.

Activation and Desensitization of Rat A3-Adenosine Receptors by Selective Adenosine Derivatives and Xanthine-7-Ribosides.

Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV Xanthine and adenosine derivatives, known to bind to recombinant rat A3 adenosine receptors stably expressed in Chinese hamster ovary cells, were characterized in a functional assay consisting of activation of A3 receptor-stimulated binding of [35S]GTPγS in rat RBL-2H3 cell membranes. 1,3-Dibutylxanthine-7-riboside-5'-N-methylcarboxamide (DBXRM, 7b), previously shown to inhibit adenylyl cyclase via rat A3 receptors with full efficacy, appeared to be a partial agonist at the rat A3 receptor of RBL-2H3 cells. Full agonists, such as Cl-IB-MECA or I-AB-MECA, were more potent and effective than the partial agonist DBXRM in causing desensitization of rat A3 receptors, as indicated by loss of [35S]GTPγS binding. At A1 receptors, antagonism of agonist-elicited inhibition of rat adipocyte adenylyl cyclase was observed for several xanthine-7-riboside derivatives that had been shown to be full agonists at rat A3 receptors. A new xanthine riboside (3'-deoxyDBXRM, 7c) was synthesized and found to be a partial agonist at rat A3 receptors and an antagonist at rat A1 receptors. Thus, it is possible for the same compound to stimulate one adenosine receptor subtype (A3) and block another subtype (A1) within the same species.

Induction of multiple effects on adenylyl cyclase regulation by chronic activation of the human A3 adenosine receptor.

The A3 adenosine receptor (A3AR) contributes to several cardiovascular effects of adenosine, including antihypertensive and cardioprotective effects. Although several studies have detailed the mechanisms underlying agonist-mediated desensitization of the rat A3AR, the regulation of the human A3AR, which displays only a 70% amino acid identity with the rat homologue, has not been addressed. Using a Chinese hamster ovary cell line stably expressing a recombinant human A3AR, we demonstrated that prolonged treatment with the AR agonist 5'-N-ethylcarboxamidoadenosine induces uncoupling of the A3AR from G proteins and functional desensitization. In addition to A3AR desensitization, a 1.5-2.5-fold increase was noted in the adenylyl cyclase (AC) activity achieved in the presence of GTP with or without forskolin. This sensitization of AC activity was not a consequence of the down-regulation of Gi proteins induced by NECA treatment and was not associated with sustained or transient increases in the expression of Gs. Time course experiments revealed that the onset of sensitization was half-maximal between 2 and 3 hr but was not due to the synthesis of new proteins because cycloheximide treatment failed to inhibit sensitization. The inability of the sensitization process to alter the AC activity obtained in the presence of manganese chloride suggests that prolonged A3AR activation increases the coupling efficiency between Gs and AC catalytic units. This phenomenon has implications for long term cellular adaptation to agonist because in agonist-treated cells, the extent to which a suboptimal concentration of forskolin could increase phosphorylation of the cAMP-responsive element binding protein was elevated compared with vehicle-treated controls.

RADIOLABELING AND EFFICIENT SYNTHESIS OF TRITIATED 2-CHLORO-N6-(3-IODOBENZYL)ADENOSINE-5'-N-METHYLURON-AMIDE, A POTENT, SELECTIVE A3 ADENOSINE RECEPTOR AGONIST.

We recently reported that 2-substitution of N6-benzyladenosine-5'-uronamides greatly enhances selectivity of agonists for rat A3 adenosine receptors J. Med. Chem.1994, 37, 3614-3621). Specifically, 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (2-CI-IB-MECA), which displayed a K1 value of 0.33 nM, is the most selective for A3 receptors yet reported with selectivity versus A1 and A2a receptors of 2500- and 1400-fold, respectively. In order to obtain pharmacological tools for the study of A3 adenosine receptors, two routes for radiolabeling of 2-CI-IB-MECA through incorporation of tritium at the 5'-methylamido group were compared. One route formed a 2',3'-protected nucleoside 5'-carboxylic acid (9), which was condensed with methylamine and deprotected. The more efficient synthesis started from D-ribose and provided 2-CI-IB-MECA (12) in six steps with an overall yield of 5.6 %. Tritium was introduced in the penultimate step by heating N6-(3-iodobenzyl)-2-chloro-2',3'-di-O-acetyl-5'-(methoxycarbonyl)adenosine (17) with [3H]methylamine in methanol at 60 °C for 2 h. The specific activity of [3H]2-CI-IB-MECA was 29 Ci/mmol with a radiochemical purity of 99%.

CDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant

A cDNA encoding a variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full-length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gl proteins Analysis of receptor transcripts by RT-PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA Identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors.

Agonist-dependent Phosphorylation and Desensitization of the Rat A3 Adenosine Receptor: EVIDENCE FOR A G-PROTEIN-COUPLED RECEPTOR KINASE-MEDIATED MECHANISM

A3 adenosine receptor (A3AR) activation contributes to both the cardioprotective and antihypertensive effects of adenosine. To date, no studies have examined the mechanisms by which this receptor undergoes rapid homologous desensitization. Therefore, a functional hemagglutinin epitope-tagged A3AR has been stably expressed in Chinese hamster ovary cells, and its regulation by the AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) has been studied. Cellular exposure to NECA induces rapid (t1/2 = approximately 1 min) A3AR phosphorylation on serine and threonine residues. This is associated with a functional desensitization and a 30-40% reduction in the number of high affinity agonist binding sites as determined by radioligand binding assays. Activation of second messenger-regulated kinases could not mimic the effect of NECA, suggesting a role for G-protein-coupled receptor kinases (GRKs). In vitro phosphorylation assays demonstrate that phosphorylation of agonist-occupied A3ARs is enhanced by GRK2 and that cellular pretreatment with NECA dramatically inhibits subsequent GRK2-mediated phosphorylation in vitro. Therefore, the A3AR is phosphorylated in situ by a kinase similar or identical to GRK2, and this may be involved in rapid functional desensitization of the A3AR.

Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors.

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

Identification of an adenosine receptor domain specifically involved in binding of 5'-substituted adenosine agonists.

The bovine A1 adenosine receptor (A1AR) and rat A3 adenosine receptor (A3AR) display distinct agonist and antagonist binding properties. To identify regions involved in ligand recognition, A1AR/A3AR chimeric receptors were created, expressed in COS-7 cells, and analyzed by radioligand binding. A chimeric receptor in which the third intracellular loop of the A1AR was replaced with that of the A3AR bound agonists and the antagonist, [3H]xanthine amine congener, with affinities identical to wild-type A1AR. A chimeric receptor with the fifth transmembrane domain (TM5) and third intracellular loop of the A1AR replaced with that of the A3AR displayed antagonist affinity similar to wild-type A1AR. However, relative to the A1AR, this chimeric demonstrated much greater affinity for 5'-substituted adenosine analogs, whereas affinity for N6-substituted compounds was unaffected. Substitution of a 6-amino acid cassette of the exofacial half of TM5 of the A3AR into the A1AR produced enhanced binding of exclusively a 5'-substituted analog, indicating involvement of this specific region in ligand recognition. These findings suggest that the 5'- and N6-substituents of adenosine agonists bind to distinct regions of ARs and that TM5 of the A3AR interacts more favorably with 5'-substituted compounds than does that of the A1AR.

CDNA cloning and sequence analysis of the human A3 adenosine receptor

We report the cloning of the cDNA encoding the homolog of the A3 adenosine receptor (A3AR) from a human heart library. The receptor shares a surprisingly low degree of sequence homology to the rat A3AR. Northern analysis of various human tissues showed the human A3AR gene to be expressed primarily in lung, liver, kidney and heart, with very little expression in the brain or in skeletal muscle.