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1.  High Affinity Acylating Antagonists for the A1 Adenosine Receptor: Identification of Binding Subunit 
Molecular pharmacology  1988;34(6):724-728.
Two isomenc isothiocyanate derivatives of the A1 adenosine receptor antagonist xanthine amine cogener (XAC) have been synthesized and found to be potent affinity labels (irreversibly bound ligands) for A1 adenosine receptors. The interaction of m- and p-isomers of 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl(2-aminoethylaminocarbonyl(4-methyloxy(phenyl)))))-xanthine (DITC-XAC) with rat brain A1 receptors is of high affinity (EC50 = 27 and 52 nm, respectively) as determined by radioligand competition curves. These compounds reduced the number of A1 receptors (>90% at 500 nm m-DITC-XAC) in brain membranes, without any change in the affinity of the remaining receptors for [125I]N6-2-(4-aminophenyl)ethyladenosine. Prior reaction of the isothiocyanate moiety with ethylenediamine did not alter the affinity of the XAC derivative for the A1 receptor but eliminated its ability to covalently incorporate into the receptor. Incubation of brain membranes with radiolabeled p- and m-DITC-XAC results in the specific labeling of a Mr 38,000 peptide. This labeling can be blocked with both an A1 adenosine receptor-specific agonist and an antagonist. This specific protein has the same molecular weight as the protein labeled with A1-selective photoaffinity probes. The much higher efficiency of incorporation of these affinity probes compared with photoaffinity probes should make them extremely useful for structural studies of A1 adenosine receptors.
PMCID: PMC3557832  PMID: 3200248
2.  125I-4-(2-[7-Amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol, a High Affinity Antagonist Radioligand Selective for the A2a Adenosine Receptor 
Molecular pharmacology  1995;48(6):970-974.
The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}-{1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5′-N-carboxamidoadenosine, 5′-N-ethylcarboxamidoadenosine, and (−)-N6-[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM241385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM241385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.
PMCID: PMC3479638  PMID: 8848012
3.  The A2 Adenosine Receptor: Guanine Nucleotide Modulation of Agonist Binding Is Enhanced by Proteolysis 
Molecular pharmacology  1991;39(2):130-135.
Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125l-2-[4-(2-{2-[(4-ammnophenyl)methylcarbonylamino]ethylaminnocarbonyl}ethyl)phenyl]ethylamino-5′-N-ethylcarboxamidoadenosine (1251-PAPA-APEC) and its photoaffinity analog 125l-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is Iabeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125l-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr ~47,O00 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory Gs-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit stnatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5′-(β,γ-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein.
Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the Gs protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.
PMCID: PMC3463105  PMID: 1899902
4.  Glycoprotein Nature of the A2-Adenosine Receptor Binding Subunit 
Molecular pharmacology  1990;38(2):177-183.
Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by covalent labeling with the photoaffinity cross-liking ligand 125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonyl-amino]ethylaminocarbonyl]ethyl]phenyl]ethylamino-5′-N-ethyl-carboxamidoadenosine or directly with the azide derivative described in this paper. The protein comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding subunit from bovine brain were compared by partial peptide maps and following treatment with exo- and endoglycosidases. Peptide maps using two separate proteases reveal that the A1- and A2-adenosine receptor binding subunits share no common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000 without intermediate peptides, suggesting a single N-linked carbohydrate chain. The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an increased mobility on gel electrophoresis, suggesting the receptors contain a glycan component containing terminal sialic acid. Treatment of the labeled A2AR with α-mannosidase reveals two distinct populations of A2ARs, one of which is sensitive and the other resistant to the enzyme. The nonadditivity of sequential treatments with the two exoglycosidases suggests, a heterogeneous population of A2AR containing either complex- or high mannose-type carbohydrate chains. These data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate chain of either the complex or high mannose type. In addition, the A1- and A2ARs are distinct glycoproteins, as evidenced by their differing molecular weights (before and after deglycosylation) and distinct peptide maps.
PMCID: PMC3454504  PMID: 2385230
5.  Structure–Activity Relationships of N6-Benzyladenosine-5′-uronamides as A3-Selective Adenosine Agonists† 
Journal of medicinal chemistry  1994;37(5):636-646.
Adenosine analogues modified at the 5′-position as uronamides and/or as N6-benzyl derivatives were synthesized. These derivatives were examined for affinity in radioligand binding assays at the newly discovered rat brain A3 adenosine receptor and at rat brain A1 and A2a receptors. 5′-Uronamide substituents favored A3 selectivity in the order N-methyl > N-ethyl ∞ unsubstituted carboxamide > N-cyclopropyl. 5′-(N-Methylcarboxamido)-N6-benzyladenosine was 37–56-fold more selective for A3 receptors. Potency at A3 receptors was enhanced upon substitution of the benzyl substituent with nitro and other groups. 5′-N-Methyluronamides and N6-(3-substituted-benzyl)adenosines are optimal for potency and selectivity at A3 receptors. A series of 3-(halobenzyl)-5′-N-ethyluronamide derivatives showed the order of potency at A1 and A2a receptors of I ~ Br > Cl > F. At A3 receptors the 3-F derivative was weaker than the other halo derivatives. 5′-N-Methyl-N6-(3-iodobenzyl)adenosine displayed a Ki value of 1.1 nM at A3 receptors and selectivity versus A1 and A2a receptors of 50-fold. A series of methoxybenzyl derivatives showed that a 4-methoxy group best favored A3 selectivity. A 4-sulfobenzyl derivative was a specific ligand at A3 receptors of moderate potency. An aryl amino derivative was prepared as a probe for radioiodination and receptor cross-linking.
PMCID: PMC4474279  PMID: 8126704
6.  A role for central A3-adenosine receptors Mediation of behavioral depressant effects 
FEBS letters  1993;336(1):57-60.
The behavioral effects of a selective A3 adenosine receptor agonist 3-IB-MECA (N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high-affinity Aragonist radioligand [125I]AB-MECA (N6-(3-iodo-4-aminobenzyl)-5′-N-methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the Kd value for binding to A3 receptors was 1.39 ± 0.04 nM with a Bmax of 14.8 ± 2.1 fmol/mg protein. 3-IB-MECA at 0.1 mg/kg i.p. was a locomotor depressant with> 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3-IB-MECA were unaffected. 3-IB-MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.
PMCID: PMC4287251  PMID: 8262217
Adenosine receptor; Xanthine; Locomotor activity; Histamine; Radioligand binding
Pharmacology communications  1992;1(2):145-154.
A new xanthine (adenosine antagonist) radioligand that binds covalently to A1-adenosine receptors was prepared and used as a receptor probe. BH-DITC-XAC was synthesized via a trifunctional aryl diisothiocyanate crosslinker. containing the p-hydroxyphenylpropionyl group for radioiodination. The xanthine competed against agonist or antagonist A1 receptor radioligands in bovine brain membranes with an IC50, of 40nM. 125I-BH-DITC-XAC, prepared directly by the chloramine T method and purified by HPLC. bound specifically to A1 receptors. This binding was inhibited in the presence of the adenosine agonists R-PIA, S-PIA. and NECA in a dose dependent manner and with the order of potency characteristic of bovine A1 receptors. Incubation of affinity purified bovine A1-receptors with 125I-BH-DITC-XAC (0.8 nM) for 2 hours resulted in the specific and clean labelling of a polypeptide band corresponding to MW 36,000, identical to that previously found for the A1 receptor.
PMCID: PMC4217529  PMID: 25374448
xanthines; adenosine receptors; affinity labeling; radioiodination
9.  A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor 
Molecular pharmacology  1994;45(6):1101-1111.
A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.
PMCID: PMC3479652  PMID: 8022403
10.  A New High Affinity, Iodinated Adenosine Receptor Antagonist as a Radioligand/Photoaffinity Crosslinking Probe 
Molecular pharmacology  1987;32(1):184-188.
A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenyl-acetylamino)ethyl]amino]carbonyl]-methyl]oxy]phenyl]-1,3-di-propylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD= 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125IPAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA > S-PIA > NECA ≫ IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.
PMCID: PMC3478951  PMID: 3614192
11.  Demonstration of Distinct Agonist and Antagonist Conformations of the A1 Adenosine Receptor* 
The Journal of biological chemistry  1989;264(22):13157-13164.
A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity probes all labeled a Mr 38,000 protein which is the A1 receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit.
Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease.
These data suggest that agonist and antagonist photoaffinity probes interact with and incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.
PMCID: PMC3478959  PMID: 2753906
12.  Purification and Characterization of Bovine Cerebral Cortex A1 Adenosine Receptor1 
A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R- phenylisopropyladenosine (R-PIA) > N6-S-phenylisopropyladenosine > (S-PIA) > 5′-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA > NECA > S-PIA.
The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy] - phenyl]-1,3-dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand ([3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax > 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA > S-PIA > NECA. The overall yield from soluble preparation was 7%.
The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to α-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.
PMCID: PMC3476062  PMID: 2275555
13.  Structure–Activity Relationships of 1,3-Dialkylxanthine Derivatives at Rat A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(20):3373-3382.
1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.
PMCID: PMC3471218  PMID: 7932565
14.  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
15.  Cloning, Expression, and Characterization of the Unique Bovine A1 Adenosine Receptor 
The Journal of biological chemistry  1992;267(15):10764-10770.
The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) > N6-S-phenylisopropyladenosine (S-PIA) > 5′-N-ethylcarboxamidoadenosinea nd has a 5–10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2–4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR
The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by >90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.
PMCID: PMC3463104  PMID: 1587851
16.  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
18.  Molecular Characterization of A1 and A2a Adenosine Receptors 
Drug development research  2004;28(3):226-231.
Detailed amino acid sequence analyses of A1 and A2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A1 and A2a receptors have been introduced. For example, an isothiocyanate, p-DITC-APEC (100 nM), irreversibly diminished the Bmax for [3H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5’-N-ethylcarboxamidoadenosine) binding in rabbit striatal membranes by 71% (Kd unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A2a receptors were characterized using [3H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-l,3-dipropylxanthine), respectively. The inhibition of A2 radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A1 receptors in which either His251 or His278 residues have been substituted with Leu suggest that both histidines are important in binding.
PMCID: PMC3446818  PMID: 23002320
affinity labeling; sequence analysis; xanthines; chemical modification; mutagenesis
19.  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
20.  Electrophilic Derivatives of Purines as Irreversible Inhibitors of A1 Adenosine Receptors 
Journal of medicinal chemistry  1989;32(5):1043-1051.
Functionalized congeners derived from 1,3-dipropyl-8-phenylxanthine and from N6-phenyladenosine were derivatized to contain electrophilic groups (isothiocyanate, N-hydroxysuccinimide ester, maleimide, sulfonyl chloride, or α-haloacyl group) capable of reaction with nucleophiles on biopolymers. The goal was to inhibit chemically the A1 adenosine receptor by using reactive agonist and antagonist ligands. Some of the electrophilic derivatives were synthesized through acylation of amine-functionalized congeners using hetero- or homobifunctional reagents available for protein cross-linking. The affinity for A1 adenosine receptors was evaluated in competitive binding assays by using rat and bovine brain membranes. Several xanthine and adenosine thiourea derivatives prepared from 1,3- and l,4-phenylene diisothiocyanate (DITC) were potent irreversible inhibitors of adenosine receptors. Derivatives of m-DITC, at concentrations between 10 and 500 nM, irreversibly eliminated binding at 90% of the A1-receptor sites. Receptor affinity of both xanthine and adenosine derivatives containing distal phenylthiourea substituents was diminished by electron-donating groups on the ring.
PMCID: PMC3442263  PMID: 2709373
21.  Role of the Second Extracellular Loop of Adenosine Receptors in Agonist and Antagonist Binding 
The Journal of biological chemistry  1994;269(40):24692-24698.
Adenosine receptor (AR) agonists and antagonists are ~100-fold and 100,000-fold, respectively, more potent at the bovine A1AR as compared to the rat A3AR. To determine regions of ARs involved in ligand recognition, chimeric receptors composed of bovine A1AR and rat A3AR sequence were constructed and their ligand binding properties examined following expression in COS-7 cells. Substitutions oft he second extracellular loop or a region encompassing transmembrane domains 6 and 7 of the A1AR into the A3AR resulted in enhanced affinities of both agonists and antagonists compared to wild-type A3AR. The region of the second extracellular loop of the A1AR responsible for this effect was identified as the distal eleven amino acids of the loop. Replacement of this segment of the A3AR with that of the A1AR in combination with the regions encompassing transmembrane domains 6 and 7 resulted in a 50,000-fold increase in the Kd for antagonist radioligand, [3H]1,3-dipropyl-8-cyclopentylxanthine. Agonist affinity at this chimeric was over 100-fold greater than that displayed by wild-type A3AR. Thus, multiple regions of ARs including a segment of the second extracellular loop are involved in ligand recognition, and considerable overlap exists in structural features required for agonist and antagonist binding.
PMCID: PMC3437327  PMID: 7929142
22.  Pharmacological Characterization of Novel A3 Adenosine Receptor-selective Antagonists 
Neuropharmacology  1997;36(9):1157-1165.
The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazo-line MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP-γ-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-α formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 μM. Published by Elsevier Science Ltd.
PMCID: PMC3433714  PMID: 9364471
Dihydropyridine; flavonoid; triazoloquinazoline; adenylate cyclase; tumor necrosis factor; guanine nucleotides; adenosine A3 receptor; adenosine
23.  Chemical Modification and Irreversible Inhibition of Striatal A2a Adenosine Receptors 
Molecular pharmacology  1992;42(1):123-133.
The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mm, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mm hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at >5 mm inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was >50 mm. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nm) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.
PMCID: PMC3429947  PMID: 1635550
Journal of receptor research  1992;12(2):149-169.
The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8±1.1 nM and a Bmax of 313± 10 fmol/mg protein. The addition of 100 μM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6±1.0 nM and 185± 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98±0.54 nM and a Bmax of 0.71±0.23 pmol/mg protein (25°C, non-specific binding defined with 100 μM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A > XAC ≈ PD115,199 > PAPA-XAC > CPX > HTQZ ≈ XCC ≈ CP-66,713 > theophylline ≈ caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.
PMCID: PMC3429337  PMID: 1583620
25.  Trifunctional Agents as a Design Strategy for Tailoring Ligand Properties: Irreversible Inhibitors of A1 Adenosine Receptors† 
Bioconjugate chemistry  1991;2(2):77-88.
The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-l,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H]CGS21680 (2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]adenosine-5′-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversibly binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.
PMCID: PMC3427756  PMID: 1868116

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