Species differences and mechanism of action of A3 adenosine receptor allosteric modulators.

Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5'-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils.

The formation of adenosine dampens inflammation by inhibiting most cells of the immune system. Among its actions on neutrophils, adenosine suppresses superoxide generation and regulates chemotactic activity. To date, most evidence implicates the G(s) protein-coupled A(2A) adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A(2B)AR is now known to be expressed in neutrophils and that it is a G(s) protein-coupled receptor, we examined in this study whether it signals to suppress neutrophil activities by using 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY 60-6583), a new agonist for the human A(2B)AR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A(2B)AR. We found that treating mouse neutrophils with low concentrations (10(-9) and 10(-8) M) of BAY 60-6583 inhibited formylated-methionine-leucine-phenylalanine (fMLP)-stimulated superoxide production by either naive neutrophils, tumor necrosis factor-α-primed neutrophils, or neutrophils isolated from mice treated systemically with lipopolysaccharide. This inhibitory action of BAY 60-6583 was confirmed to involve the A(2B)AR in experiments using neutrophils obtained from A(2B)AR gene knockout mice. It is noteworthy that BAY 60-6583 increased fMLP-stimulated superoxide production at higher concentrations (>1 µM), which was attributed to an AR-independent effect. In a standard Boyden chamber migration assay, BAY 60-6583 alone did not stimulate neutrophil chemotaxis or influence chemotaxis in response to fMLP. These results indicate that the A(2B)AR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A(2A)AR in regulating the proinflammatory actions of neutrophils.

Polyamidoamine (PAMAM) dendrimer conjugate specifically activates the A3 adenosine receptor to improve post-ischemic/reperfusion function in isolated mouse hearts.

BACKGROUND: When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR. RESULTS: A PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific. CONCLUSIONS: Covalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.

Structure-Based Design, Synthesis by Click Chemistry and in Vivo Activity of Highly Selective A3 Adenosine Receptor Agonists.

2-Arylethynyl derivatives of (N)-methanocarba adenosine 5'-uronamides are selective A3AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabolic liability. Docking of nucleosides containing possible short linker moieties at the adenine C2 position using a hybrid molecular model of the A3AR (based on the A2AAR agonist-bound structure) correctly predicted that a triazole would maintain the A3AR selectivity, due to its ability to fit a narrow cleft in the receptor. The analogues with various N6 and C2-aryltriazolyl substitution were synthesized and characterized in binding (Ki at hA3AR 0.3 - 12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain (chronic constriction injury). Among N6-methyl derivatives, a terminal pyrimidin-2-yl group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo. N6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85% protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, >90% protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined structure-based methods and phenotypic screening to identify nucleoside derivatives having translational potential.

In vivo phenotypic screening for treating chronic neuropathic pain: modification of C2-arylethynyl group of conformationally constrained A3 adenosine receptor agonists.

(N)-Methanocarba adenosine 5'-methyluronamides containing 2-arylethynyl groups were synthesized as A3 adenosine receptor (AR) agonists and screened in vivo (po) for reduction of neuropathic pain. A small N(6)-methyl group maintained binding affinity, with human > mouse A3AR and MW < 500 and other favorable physicochemical properties. Emax (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A3AR agonist, 2-(3,4-difluorophenylethynyl)-N(6)-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding Ki, hA3AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed particularly long in vivo duration (>3 h). Selected analogues were docked to an A3AR homology model to explore the environment of receptor-bound C2 and N(6) groups. Various analogues bound with µM affinity at off-target biogenic amine (M2, 5HT2A, ß3, 5HT2B, 5HT2C, and α2C) or other receptors. Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment.

Adenosine A1 receptors heterodimerize with ß1- and ß2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling.

G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and ß-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with ß1- and/or ß2-adrenergic receptors (ß1R and ß2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, ß1, and ß2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both ß1R and ß2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both ß1R and ß2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/ß1R and A1AR/ß2R heteromers. ß-Receptor-mediated cAMP production was not altered in A1AR/ß1R expressing cells, but was significantly reduced in the A1AR/ß2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/ß1R and A1AR/ß2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, ß1R, and ß2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and ß receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.

Characterization by flow cytometry of fluorescent, selective agonist probes of the A(3) adenosine receptor.

Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.

Rational design of sulfonated A3 adenosine receptor-selective nucleosides as pharmacological tools to study chronic neuropathic pain.

(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood-brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N(6)-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N(6)-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine's CNS vs peripheral actions.

Activation of the A(3) adenosine receptor inhibits fMLP-induced Rac activation in mouse bone marrow neutrophils.

Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A(2A)AR, we have recently shown that selective activation of the abundantly expressed A(3)AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A(3)AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A(3)AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A(3)AR agonist CP-532,903 reduced fMLP-induced Rac activation using an ELISA-based assay that detects all three Rac isoforms. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but not activation of ERK1/2 or p38. Pre-treating neutrophils with CP-532,903 did not stimulate cAMP production or alter fMLP-induced calcium transients, implicating that A(3)AR stimulation does not inhibit Rac activation or neutrophil activities by suppressing Ca(2+) signaling, elevating the intracellular concentration of cAMP, or by cross-desensitizing fMLP receptors. Our results suggest that activation of the A(3)AR signals to suppress neutrophil functions by interfering with the monomeric GTPase Rac, thus contributing to the ant-inflammatory actions of adenosine.

Synthesis and pharmacological characterization of [(125)I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor.

A recently reported selective agonist of the human A(3) adenosine receptor (hA(3)AR), MRS5127 (1'R,2'R,3'S,4'R,5'S)-4'-[2-chloro-6-(3-iodobenzylamino)-purine]-2',3'-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5'-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A(3)ARs. The radioiodination of the N(6)-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [(125)I]MRS5127 bound to the human A(3)AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K(d) value of 5.74+/-0.97nM. At a concentration equivalent to its K(d), non-specific binding comprised 27+/-2% of total binding. In kinetic studies, [(125)I]MRS5127 rapidly associated with the hA(3)AR (t(1/2)=0.514+/-0.014min), and the affinity calculated from association and dissociation rate constants was 3.50+/-1.46nM. The pharmacological profile of ligands in competition experiments with [(125)I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA(3)AR. [(125)I]MRS5127 bound with similar high affinity (K(d), nM) to recombinant A(3)ARs from mouse (4.90+/-0.77), rabbit (2.53+/-0.11), and dog (3.35+/-0.54). For all of the species tested, MRS5127 exhibited A(3)AR agonist activity based on negative coupling to cAMP production. Thus, [(125)I]MRS5127 represents a new species-independent agonist radioligand for the A(3)AR. The major advantage of [(125)I]MRS5127 compared with previously used A(3)AR radioligands is its high affinity, low degree of non-specific binding, and improved A(3)AR selectivity.