His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3′ position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3′-ureido, but not 3′-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2′- and 3′-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3′-ureido-3′-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3′-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2′- and 3′-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5′ position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.
Adenosine A3 receptors are of interest in the treatment of cardiac ischemia, inflammation, and neurodegenerative diseases. In an effort to create a unique receptor mutant that would be activated by tailor-made synthetic ligands, we mutated the human A3 receptor at the site of a critical His residue in TM7, previously proposed to be involved in ligand recognition through interaction with the ribose moiety. The H272E mutant receptor displayed reduced affinity for most of the uncharged A3 receptor agonists and antagonists examined. For example, the nonselective agonist 1a was 19-fold less potent at the mutant receptor than at the wild-type receptor. The introduction of an amino group on the ribose moiety of adenosine resulted in either equipotency or enhanced binding affinity at the H272E mutant relative to wild-type A3 receptors, depending on the position of the amino group. 3′-Amino-3′-deoxyadenosine proved to be 7-fold more potent at the H272E mutant receptor than at the wild-type receptor, while the corresponding 2′- and 5′-amino analogues did not display significantly enhanced affinities. An 3′-amino-N6-iodobenzyl analogue showed only a small enhancement at the mutant (Ki = 320 nM) vs wild-type receptors. The 3′-amino group was intended for a direct electrostatic interaction with the negatively charged ribose-binding region of the mutant receptor, yet molecular modeling did not support this notion. This design approach is an example of engineering the structure of mutant receptors to recognize synthetic ligands for which they are selectively matched on the basis of molecular complementarity between the mutant receptor and the ligand. We have termed such engineered receptors “neoceptors”, since the ligand recognition profile of such mutant receptors need not correspond to the profile of the parent, native receptor.
Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A2A adenosine receptor (AR). Adenosine derivatives functionalized at the 5′, 2, and N6 positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5′-(2-aminoethyl)uronamide but not 5′-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N6-(2-methylbenzyl) group enhanced affinity at the Q89D- and N181D- but not the T88D-A2AAR. Amino groups placed near the 2- or N6-position only slightly affected the binding to mutant receptors. The 5′-hydrazide MRS3412 was 670-and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A2AAR compared to the wild-type. Thus, we identified and modeled pairs of A2AAR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.
Efforts to model and reengineer the putative binding sites of G protein-coupled receptors (GPCRs) have led to an approach to combining small molecule “classical” medicinal chemistry and gene therapy. By this approach, complementary structural changes, for example, based on novel ionic or H bonds, are made in the receptor and ligand for selective enhancement of affinity. Thus, a modified receptor (neoceptor) is designed for activation by tailor-made agonists that do not interact with the native receptor. The neoceptor is no longer activated by the native agonist, but rather acts as scaffold for docking of novel small molecules (neoligands). In theory, the approach could verify the accuracy of GPCR molecular modeling, dissection of signaling, design of small molecules to rescue disease-related mutations, and small-molecule-directed gene therapy. The neoceptor-neoligand pairing may offer spacial specificity by delivering the neoceptor to a target site and temporal specificity by administering neoligand when needed.
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-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-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.
An integrated approach to the study of drug-receptor interactions has been applied to adenosine receptors (ARs) and P2Y nucleotide receptors. This approach includes probing the receptor structure through site-directed mutagenesis and molecular modeling, in concert with altering the structure of the agonist ligands. Goals of this structural approach are to generate a testable hypothesis for location of the binding site and subsequently to enable the rational design of new agonists and antagonists. In this manner, receptor subtype selectivity has been increased, and agonists have been converted into partial agonists and antagonists. An approach to receptor engineering (neoceptors) has been explored, in which synthetic small molecule agonists (neoligands) are specifically tailored to activate only receptors in which the putative binding sites have been modified. This orthogonal approach to receptor activation, intended for eventual gene therapy, has been demonstrated for A3 and A2A ARs.
Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5′-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem.
37, 636–646), were synthesized starting from methyl β-d-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5′-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na+-independent adenosine transporter, as indicated in displacement of [3H]N6-(4-nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5′-position and a 3-iodobenzyl group at the N6-position.
Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A1 and A3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
The highly selective agonists of the A3 adenosine receptor (AR), Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) and its 4′-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5′-uronamide position. The 2-chloro-5′-(N,N-dimethyl)uronamido analogues bound to, but did not activate the human A3AR, with Ki values of 29 nM (4′-O) and 15 (4′-S) nM, showing >100-fold selectivity over A1, A2A, and A2BARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5′-(N,N-dimethyl)uronamido substitution also retained A3AR selectivity but lowered affinity.
nucleoside; G protein-coupled receptor; adenylyl cyclase; molecular modeling; radioligand binding; AR, adenosine receptor; CGS21680, 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine; CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine; CPA, N6-cyclopentyladenosine; DMEM, Dulbecco’s modified Eagle’s medium; I-AB-MECA, N6-(4-amino-3-iodobenzyl)-5′-N-methylcarboxamidoadenosine; NECA, 5′-N-ethylcarboxamidoadenosine; PIA, N6-(phenylisopropyl)adenosine; PTLC, preparative thin layer chromatography
The objective of this study was to create constitutively active mutant human A3 adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A3 ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A3 agonist Cl-IB-MECA (2–chloro-N6-(3–iodobenzyl)adenosine-5′-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC50 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F and Y282F mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.
purines; G protein-coupled receptor; phospholipase C; adenylyl cyclase; radioligand binding; nucleosides
Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A1 and possibly A3 receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A1 adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1 μM), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A3 adenosine receptor agonist N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA, 0.1 μM), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca2+ that lasted 40–60 sec. Pretreatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca2+ elevation. In conclusion, activation of adenosine A1 receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A3 receptors confers partial cardioprotection.
A1 and A3 adenosine receptors; angiotensin II; apoptosis; cardiomyocytes; Feulgen and TUNEL stainings
The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2′-, 3′-, and 5′-deoxy; 2′- and 3′-O-methyl; 2′-deoxy 2′-fluoro; 6′-thio; 5′-uronamide; carbocyclic; 4′- or 3′-methyl; and inversion of configuration). (−)- and (+)-5′-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6′-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(β-L-2′-deoxy-6′-thiolyxofuranoside) displayed a Ki value of 8 μM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropylxanthine). 2-Chloro-2′-deoxyadenosine and 2-chloroadenin-9-yl(β-D-6′-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 μM, respectively. The A2a selective agonist 2-(1-hexynyl)-5′-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4′-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4′-methyladenosine-5′-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.
We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2′- and 3′-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2′-OH being more essential. Thus, the 2′-fluoro substitution eliminated both binding and activation, while a 3′-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5′-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3′-fluoro derivatives. The 4′-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5′-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1′- to 4′-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a KB value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
A variety of adenosine analogs activate phosphoinositide breakdown in a rat RBL-2H3 mast cell line. It is presumed that an A3-adenosine receptor is involved, since the phosphoinositide response is insensitive to xanthines. However, the very potent A3- receptor agonist 2-chloro-N6-iodobenzyl-N-methylcarboxamidoadenosine (2-CI-IBMECA) with an EC50 of 4.1 µM is about twofold less potent (and less efficacious) than N-ethylcarboxamidoadenosine (NECA) with an EC50 of 2.1 µM. The other agents consisting of N6-p-aminophenylethyladenosine (APNEA), N6-iodobenzylMECA (IB-MECA), N6-R- phenylisopropyladenosine (R-PIA), 2-chloroadenosine, N6-benzyladenosine, N6- cyclohexyladenosine (CHA), N6-cyclohexylNECA (CHNECA), 2-(p- carboxyethylphenyl-ethylaminoNECA (CGS 21680), 1,3-dibutylxanthine 7-riboside-5′-N-methylcarboxamide (DBXRM), adenosine, and 8-bromoadenosine are all nearly equipotent with EC50 values of 5.5-13.9 µM. The rank order of potencies of the analogs in causing an elevation of intracellular calcium is quite different. The potent A3 receptor agonists 2-CI-IBMECA and IB-MECA with EC50 values of 0.07 and 0.11 µM, respectively, are about fourfold more potent than N6-cyclohexylNECA and about 15-fold more potent than NECA. The other analogs are comparable or somewhat less potent than NECA, some are less efficacious, and 8-bromoadenosine is inactive. The results suggest that stimulation of phosphoinositide breakdown by adenosine analogs in RBL-2H3 cells as measured by IP1 accumulation is not predictive of IP3-mediated elevations of intracellular calcium. Rank order of potency for the calcium response is consonant with intermediacy of A3-adenosine receptors, while the former, as measured by [3H]IP1-formation, probably reflects contributions from both an A3-mediated response and some other mechanism. Combinations of subthreshold concentrations of 2-CI-IBMECA with either the A1-selective agonist CHA or the A2A-selective agonist CGS 21680 caused a marked stimulation of phosphoinositide breakdown, providing further evidence for dual mechanisms. The selective A3-adenosine receptor antagonist 3,6-dichloro-2′-(isopropyloxy)-4′-methylflavone (MRS 1067) inhibits 2-CI-IBMECA- and NECA-elicited elevation of calcium levels, and had differential effects on phosphoinositide breakdown, blocking [3H]IP3 accumulation and either blocking (NECA) or having no effect (2-CI-IBMECA) on [3H]IP1 accumulation.
adenosine receptors; phosphoinositides; calcium; xanthines
The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [3H]-cyclic adenosine monophosphate ([3H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α1-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca2+ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml−1 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).In the presence of phenylephrine (1 μM), NECA and the A1 adenosine receptor selective agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC50 values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A3 adenosine receptor agonists N6-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N6-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pKB 8.75±0.88) and the maximal response to NECA was reduced.In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC50 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).(S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [3H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [3H]-cyclic AMP in preparations of epididymis (pEC50 values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [3H]-cyclic AMP was antagonised by XAC (apparent pKB 6.88±0.88) and also by the A2A adenosine receptor antagonist, ZM 241385 (apparent pKB 8.60± 0.76).These studies are consistent with the action of stable adenosine analogues at post-junctional A1 and A2 adenosine receptors in the epididymis of the guinea-pig. A1 Adenosine receptors potentiate α1-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A2 adenosine receptors, possibly of the A2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.
guinea-pig epididymis; A1 adenosine receptor; A2 adenosine receptor
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
We have studied the difference in receptor binding activity between partial and full β2-adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the β2-adrenoceptor, amino acid residues that are important for activation of the β2-adrenoceptor.In the binding study with [125I]-iodocyanopindolol, the Ki values of (±)-salbutamol, (±)-salmeterol, TA-2005 and (−)-isoprenaline for the β2-adrenoceptor expressed in COS-7 cell membranes were 3340, 21.0, 12.0 and 904 nM, respectively. The β1/β2 selectivity of these agonists was in the order of (±)-salmeterol (332 fold)>TA-2005 (52.8)>(±)-salbutamol (6.8)>(−)-isoprenaline (1.1), and the β3-/β2-adrenoceptor selectivity of these agonists was in the order of TA-2005 (150 fold)>(±)-salmeterol (88.6)>(±)-salbutamol (10.4)>(−)-isoprenaline (3.2).The maximal activation of adenylyl cyclase by stimulation of the β1-, β2- and β3-adrenoceptors by TA-2005 was 32, 100 and 100% of that by (−)-isoprenaline, respectively, indicating that TA-2005 is a full agonist at the β2- and β3-adrenoceptors and a partial agonist at the β1-adrenoceptor. (±)-Salbutamol and (±)-salmeterol were partial agonists at both β1- (8% and 9% of (−)-isoprenaline) and β2- (83% and 74% of (−)-isoprenaline) adrenoceptors.The affinities of full agonists, TA-2005 and (−)-isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the β2-adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (±)-salbutamol and (±)-salmeterol. Although the affinities of full agonists for the S207A-β2-adrenoceptor were decreased, those of partial agonists for the S207A-β2-adrenoceptor were essentially the same as for the wild type receptor.The constitutively active mutant (L266S, L272A) of the β2-adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (±)-salbutamol and (±)-salmeterol for the S207A-β2-adrenoceptor were essentially the same as those for the wild type β2-adrenoceptor, the affinities of (±)-salbutamol and (±)-salmeterol for the constitutively active β2-adrenoceptor were decreased by substitution of Ser207.These results suggest that Ser204 and Ser207 of the wild type and constitutively active β2-adrenoceptors differentially interacted with β2-selective agonists.
TA-2005; (±)-salbutamol; (±)-salmeterol; β2-adrenoceptor; subtype selectivity; adenlyl cyclase activity; full agonist; partial agonist; constitutively active receptor
Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca2+]i) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), and the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca2+]i. In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido-adenosine (100 nM) or N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (100 nM), selective agonists at adenosine A2A receptors and adenosine A3 receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP3) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A1 receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP3 signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.
adenosine A1 receptor; calcium; cAMP; human bronchial smooth muscle; insulin
Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM and cytosolic calcium concentrations ([Ca2+]i) were determined ratiometrically by imaging single cells. The non-selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca2+]i. In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A2A receptors and adenosine A3 receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP3) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A1 receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP3 signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.
Adenosine; calcium; human bronchial smooth muscle; adenosine A1 receptor; cAMP; insulin
Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia.
Ligands of the various adenosine receptor subtypes modulate the production of pro-and anti-inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1α production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP-1α production and affects the course of inflammation in collagen-induced arthritis.In the cultured macrophages, the A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA), and, less potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS; 1–200 μM) dose-dependently suppressed the production of MIP-1α. The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1–200 μM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP-1α production by the A3 and A2 agonist was associated with suppression of its steady-state mRNA levels.Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP-1α expression. Since IB-MECA was the most potent inhibitor of MIP-1α expression, we next investigated whether it affects the production of other pro-inflammatory mediators. We observed that IB-MECA (1–300 μM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages.Since MIP-α is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB-MECA affects the course of inflammation, MIP-α production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB-MECA inhibited the formation of MIP-1α, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration.We conclude that adenosine receptor agonists, most notably the A3 agonist IB-MECA suppress the production of MIP-α, and exert anti-inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.
Inflammation; cytokines; arthritis; xanthine; arthritis
To investigate the effects of adenosine on endogenous Xenopus oocyte receptors, we analysed defolliculated oocytes injected with mRNAs for the G protein-activated inwardly rectifying K+ (GIRK) channels.In oocytes injected with mRNAs for either GIRK1/GIRK2 or GIRK1/GIRK4 subunits, application of adenosine or ATP reversibly induced inward K+ currents, although ATP was less potent than adenosine. The responses were attenuated by caffeine, a non-selective adenosine receptor antagonist. Furthermore, in uninjected oocytes from the same donor, adenosine produced no significant current.The endogenous receptor was activated by two selective A1 adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and N6-cyclohexyladenosine (CHA), and antagonized by a selective A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopenylxanthine (DPCPX) at moderate nanomolar concentrations, but insensitive to micromolar concentrations of selective A2A and A3 adenosine receptor agonists, 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680) and N6-(3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine (IB-MECA), respectively. However, the pharmacological characteristics of the receptor were different from those of the cloned Xenopus A1 adenosine receptor and previously proposed adenosine receptors.The adenosine-induced GIRK currents were abolished by injection of pertussis toxin and CPA inhibited forskolin-stimulated cyclic AMP accumulation.We conclude that an adenosine receptor on the Xenopus oocyte membrane can activate GIRK channels and inhibit adenylyl cyclase via Gi/o proteins. Moreover, our results suggest the existence of an endogenous adenosine receptor with the unique pharmacological characteristics. As the receptor was activated by nanomolar concentrations of adenosine, which is a normal constituent of extracellular fluid, the receptor may be involved in some effects through the Gi/o protein signalling pathways in ovarian physiology.
Adenosine receptor; Gi/o protein; G protein-activated inwardly rectifying K+ (GIRK) channel; Xenopus oocyte
Activation of either the A1 or the A3 adenosine receptor (A1R or A3R, respectively) elicits delayed cardioprotection against infarction, ischemia, and hypoxia. Mitochondrial contribution to the progression of cardiomyocyte injury is well known; however, the protective effects of adenosine receptor activation in cardiac cells with a respiratory chain deficiency are poorly elucidated. The aim of our study was to further define the role of A1R and A3R activation on functional tolerance after inhibition of the terminal link of the mitochondrial respiratory chain with sodium azide, in a state of normoxia or hypoxia, compared with the effects of the mitochondrial ATP-sensitive K+ channel opener diazoxide. Treatment with 10 mM sodium azide for 2 h in normoxia caused a considerable decrease in the total ATP level; however, activation of adenosine receptors significantly attenuated this decrease. Diazoxide (100 µM) was less effective in protection. During treatment of cultured cardiomyocytes with hypoxia in the presence of 1 mM sodium azide, the A1R agonist 2-chloro-N6-cyclopentyladenosine was ineffective, whereas the A3R agonist 2-chloro-N6-iodobenzyl-5′-N-methylcarboxamidoadenosine (Cl-IB-MECA) attenuated the decrease in ATP level and prevented cell injury. Cl-IB-MECA delayed the dissipation in the mitochondrial membrane potential during hypoxia in cells impaired in the mitochondrial respiratory chain. In cells with elevated intracellular Ca2+ concentration after hypoxia and treatment with NaN3 or after application of high doses of NaN3, Cl-IB-MECA immediately decreased the elevated intracellular Ca2+ concentration toward the diastolic control level. The A1R agonist was ineffective. This may be especially important for the development of effective pharmacological agents, because mitochondrial dysfunction is a leading factor in the pathophysiological cascade of heart disease.
Ca2+ transience; hypoxia; ATP-sensitive K+ channel; sodium azide; heart disease; ischemia
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%.
Adenosine Derivatives; Radioligands; Adenosine Receptors; Tritium; Nucleosides
The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study.Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed.The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122±22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively.The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37±1%) in comparison to CPA (66±5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production.This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor.
Adenosine A1 receptor; allosteric modulation; LUF5831; thermodynamics; PD81,723; T277A mutant