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1.  Probing the Binding Site of the A1 Adenosine Receptor Reengineered for Orthogonal Recognition by Tailored Nucleosides 
Biochemistry  2007;46(25):7437-7448.
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.
PMCID: PMC3140710  PMID: 17542617
2.  Neoceptor Concept Based on Molecular Complementarity in GPCRs: A Mutant Adenosine A3 Receptor with Selectively Enhanced Affinity for Amine-Modified Nucleosides 
Journal of medicinal chemistry  2001;44(24):4125-4136.
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.
PMCID: PMC3413945  PMID: 11708915
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.
PMCID: PMC2495023  PMID: 17280720
4.  A Neoceptor Approach to Unraveling Microscopic Interactions between the Human A2A Adenosine Receptor and Its Agonists 
Chemistry & biology  2005;12(2):237-247.
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.
PMCID: PMC3122079  PMID: 15734651
5.  Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(10):1720-1735.
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.
PMCID: PMC3445626  PMID: 7752196
6.  Purine Receptors: GPCR Structure and Agonist Design 
Molecular interventions  2004;4(6):337-347.
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.
PMCID: PMC3418328  PMID: 15616163
7.  2-Substitution of N6-Benzyladenosine-5′-uronamides Enhances Selectivity for A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(21):3614-3621.
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. 1994, 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.
PMCID: PMC3468333  PMID: 7932588
8.  Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists 
Journal of medicinal chemistry  2000;43(11):2196-2203.
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.
PMCID: PMC3471159  PMID: 10841798
9.  Conversion of A3 Adenosine Receptor Agonists into Selective Antagonists by Modification of the 5′-Ribofuran-uronamide Moiety 
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.
PMCID: PMC1351218  PMID: 16289820
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
10.  Search for New Purine- and Ribose-Modified Adenosine Analogues as Selective Agonists and Antagonists at Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(7):1174-1188.
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.
PMCID: PMC3457658  PMID: 7707320
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.
PMCID: PMC3626079  PMID: 11396942
purines; G protein-coupled receptor; phospholipase C; adenylyl cyclase; radioligand binding; nucleosides
12.  Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety 
Biochemical pharmacology  2004;67(5):893-901.
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.
PMCID: PMC3150582  PMID: 15104242
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
13.  Activation of Phosphoinositide Breakdown and Elevation of Intracellular Calcium in a Rat RBL-2H3 Mast Cell Line by Adenosine Analogs: Involvement of A3-Adenosine Receptors? 
Drug development research  1996;39(1):36-46.
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.
PMCID: PMC3475513  PMID: 23087534
adenosine receptors; phosphoinositides; calcium; xanthines
14.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
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.
PMCID: PMC3953614  PMID: 11641434
15.  Adenosine A1 receptors mediate mobilization of calcium in human bronchial smooth muscle cells 
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.
PMCID: PMC2065849  PMID: 16709961
Adenosine; calcium; human bronchial smooth muscle; adenosine A1 receptor; cAMP; insulin
16.  Adenosine A1 Receptors Mediate Mobilization of Calcium in Human Bronchial Smooth Muscle Cells 
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.
PMCID: PMC2065849  PMID: 16709961
adenosine A1 receptor; calcium; cAMP; human bronchial smooth muscle; insulin
17.  Role of adenosine A1 and A3 receptors in regulation of cardiomyocyte homeostasis after mitochondrial respiratory chain injury 
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.
PMCID: PMC3457058  PMID: 15681707
Ca2+ transience; hypoxia; ATP-sensitive K+ channel; sodium azide; heart disease; ischemia
18.  Site-directed Mutagenesis Identifies Residues Involved in Ligand Recognition in the Human A2a Adenosine Receptor 
The Journal of biological chemistry  1995;270(23):13987-13997.
The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5–7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nm) and [3H]XAC (4 nm), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5′. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were >30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5’-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.
PMCID: PMC3427751  PMID: 7775460
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%.
PMCID: PMC3572746  PMID: 23598401
Adenosine Derivatives; Radioligands; Adenosine Receptors; Tritium; Nucleosides
20.  Anti-inflammatory effects of adenosine N1-oxide 
Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine’s extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.
The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.
ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.
Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.
PMCID: PMC4308844  PMID: 25632271
Adenosine; Anti-inflammatory effect; Pro-inflammatory cytokines; Adenosine receptor agonists; Endotoxin shock
21.  Partial Agonists for A3 Adenosine Receptors 
Selective agonists for A3 adenosine receptors (ARs) could potentially be therapeutic agents for a variety of disorders, including brain and heart ischemic conditions, while partial agonists may have advantages over full agonists as a result of an increased selectivity of action. A number of structural determinants for A3AR activation have recently been identified, including the N6-benzyl group, methanocarba substitution of ribose, 2-chloro and 2-fluoro substituents, various 2’- and 3’-substitutions and 4’-thio substitution of oxygen. The 2-chloro substitution of CPA and R-PIA led to A3 antagonism (CCPA) and partial agonism (Cl-R-PIA). 2-Chloroadenosine was a full agonist, while 2-fluoroadenosine was a partial agonist. Both 2’- and 3’- substitutions have a pronounced effect on its efficacy, although the effect of 2’-substitution was more dramatic. The 4-thio substitution of oxygen may also diminish efficacy, depending on other substitutions. Both N6-methyl and N6-benzyl groups may contribute to the A3 affinity and selectivity; however, an N6-benzyl group but not an N6-methyl group diminishes A3AR efficacy. N6-benzyl substituted adenosine derivatives have similar potency for human and rat A3ARS while N6-methyl substitution was preferable for the human A3AR. The combination of 2-chloro and N6-benzyl substitutions appeared to reduce efficacy further than either modification alone. The A2AAR agonist DPMA was shown to be an antagonist for the human A3AR. Thus, the efficacy of adenosine derivatives at the A3AR appears to be more sensitive to small structural changes than at other subtypes. Potent and selective partial agonists for the A3AR could be identified by screening known adenosine derivatives and by modifying adenosine and the adenosine derivatives.
PMCID: PMC3425644  PMID: 15078216
22.  The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice 
Purinergic Signalling  2012;9(2):207-214.
This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-012-9340-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3646123  PMID: 23242571
Adenosine receptor agonists; CPA; IB-MECA; Hematopoietic stem cells; Long-term repopulating ability
23.  Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position 
Bioorganic & medicinal chemistry  2004;12(11):2995-3007.
We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N6-substitutions known to enhance human A3AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A1, A2A, A2B, and A3ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA3AR affinity and efficacy in the cases of N6-(3-iodobenzyl) and N6-(trans-2-phenyl-1-cyclopropyl), for which a full A3AR agonist was converted into a selective antagonist; the 2-cyano-N6-methyl analogue was a full A3AR agonist. The combination of N6-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A1AR. The environment surrounding the 2-position within the putative A3AR binding site was explored using rhodopsin-based homology modeling and ligand docking.
PMCID: PMC3463103  PMID: 15142558
Purines; Cyclic AMP; Binding; Antagonists; Agonists; GPCR; Molecular modeling
24.  Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  1998;41(17):3186-3201.
The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. 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. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.
PMCID: PMC3474377  PMID: 9703464
25.  Selective A3 Adenosine Receptor Antagonists Derived from Nucleosides Containing a Bicyclo[3.1.0]hexane Ring System 
Bioorganic & medicinal chemistry  2008;16(18):8546-8556.
We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH, however these derivatives did not display the desired activity as selective A3 AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A3 AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b – 39b (N6-3-halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of 0.7 − 1.4 nM. In a functional assay of [35S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a KB of 8.9 nM. Thus, a highly potent and selective series of A3AR antagonists has been described.
PMCID: PMC2593936  PMID: 18752961
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase

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