Elizabeth Gizewski

Adenosine Suppresses Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production by Murine Macrophages through a Protein Kinase A- and Exchange Protein Activated by cAMP-Independent Signaling Pathway

Adenosine is generated during tissue hypoxia and stress, which reduces inflammation by suppressing the activity of most immune cells. Among its various actions, adenosine suppresses the production of proinflammatory cytokines including tumor necrosis factor (TNF)-α, through the cAMP-elevating A2A adenosine receptor (AR) subtype. In this study, we examined the signaling mechanisms by which A2AAR activation inhibits TNF-α production in thioglycollate-elicited mouse peritoneal macrophages. Pretreating murine macrophages with the nonselective AR agonist adenosine-5′-N-ethylcarboxamide (NECA), the A2AAR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), or the cAMP-elevating agent forskolin reduced TNF-α production in response to lipopolysaccharide (LPS) by greater than 60%. All of these agents increased cAMP production in macrophages and activated protein kinase A (PKA). However, we were surprised to find that treating macrophages with three different PKA inhibitors or small interfering RNA-mediated knockdown of the exchange protein activated by cAMP (Epac-1) failed to block the suppressive actions of NECA or forskolin on LPS-induced TNF-α release. Instead, okadaic acid was effective at low concentrations that selectively inhibit protein serine/threonine phosphatases. Subsequent studies showed that NECA and forskolin decreased LPS-induced steady-state TNF-α mRNA levels; this effect was due to a decreased rate of transcription based on assays examining the rate of generation of primary TNF-α transcripts. Treatment with NECA or forskolin did not interfere with LPS-induced translocation or DNA binding of the RelA/p65 subunit of nuclear factor-κB or phosphorylation of inhibitor of nuclear factor-κB-α, extracellular signal-regulated kinase 1/2, c-Jun NH2-terminal kinase, or p38 kinase. Our results suggest that AR activation inhibits LPS-induced TNF-α production by murine macrophages at the level of gene transcription through a unique cAMP-dependent, but PKA- and Epac-independent, signaling pathway involving protein phosphatase activity.

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 Gs protein-coupled A2A adenosine receptor (AR) as the primary AR subtype responsible for mediating the actions of adenosine on neutrophils by stimulating cAMP production. Given that the A2BAR is now known to be expressed in neutrophils and that it is a Gs 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 A2BAR that was confirmed in preliminary studies to be a potent and highly selective agonist for the murine A2BAR. 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 A2BAR in experiments using neutrophils obtained from A2BAR 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 A2BAR signals to suppress oxidase activity by murine neutrophils, supporting the idea that this low-affinity receptor for adenosine participates along with the A2AAR in regulating the proinflammatory actions of neutrophils.

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 adenosines CNS vs peripheral actions.

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 N6-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)-N6-(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 N6 groups. Various analogues bound with μM affinity at off-target biogenic amine (M2, 5HT2A, β3, 5HT2B, 5HT2C, and α2C) or other receptors. Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment.

Characterization of the A2B Adenosine Receptor from Mouse, Rabbit, and Dog

We have cloned and pharmacologically characterized the A2B adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A2BAR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A2BAR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A2BAR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A2BAR103-111). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A2BAR and seems to result from the use of an additional 5′ donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [3H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-3H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([3H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A2BARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A2BAR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A2BAR compared with A2BARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A2BAR103-111 variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A2BAR exist between species and that some species express nonfunctional variants of the A2BAR due to “leaky” splicing.

Adenosine A1 receptors heterodimerize with β1- and β2-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 β-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 β1- and/or β2-adrenergic receptors (β1R and β2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, β1, and β2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both β1R and β2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both β1R and β2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/β1R and A1AR/β2R heteromers. β-Receptor-mediated cAMP production was not altered in A1AR/β1R expressing cells, but was significantly reduced in the A1AR/β2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/β1R and A1AR/β2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, β1R, and β2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and β 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 A3 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.

Activation of the A3 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.

Rigidified A3 Adenosine Receptor Agonists: 1-Deazaadenine Modification Maintains High in Vivo Efficacy

Substitution of rigidified A3 adenosine receptor (AR) agonists with a 2-((5-chlorothiophen-2-yl)ethynyl) or a 2-(4-(5-chlorothiophen-2-yl)-1H-1,2,3-triazol-1-yl) group provides prolonged protection in a model of chronic neuropathic pain. These agonists contain a bicyclo[3.1.0]hexane ((N)-methanocarba) ring system in place of ribose, which adopts a receptor-preferred conformation. N (6)-Small alkyl derivatives were newly optimized for A3AR affinity and the effects of a 1-deaza-adenine modification probed. 1-Deaza-N (6)-ethyl alkyne 20 (MRS7144, K i 1.7 nM) and 1-aza N (6)-propyl alkyne 12 (MRS7154, K i 1.1 nM) were highly efficacious in vivo. Thus, the presence of N1 is not required for nanomolar binding affinity or potent, long-lasting functional activity. Docking of 1-deaza compounds to a receptor homology model confirmed a similar binding mode as previously reported 1-aza derivatives. This is the first demonstration in nonribose adenosine analogues that the 1-deaza modification can maintain high A3AR affinity, selectivity, and efficacy.

Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists

Purine (N)-methanocarba-5′-N-alkyluronamidoriboside A3 adenosine receptor (A3AR) agonists lacking an exocyclic amine resulted from an unexpected reaction during a Sonogashira coupling and subsequent aminolysis. Because the initial C6-Me and C6-styryl derivatives had unexpectedly high A3AR affinity, other rigid nucleoside analogues lacking an exocyclic amine were prepared. Of these, the C6-Me-(2-phenylethynyl) and C2-(5-chlorothienylethynyl) analogues were particularly potent, with human A3AR Ki values of 6 and 42 nM, respectively. Additionally, the C2-(5-chlorothienyl)-6-H analogue was potent and selective at A3AR (MRS7220, Ki 60 nM) and also completely reversed mouse sciatic nerve mechanoallodynia (in vivo, 3 μmol/kg, po). The lack of a C6 H-bond donor while maintaining A3AR affinity and efficacy could be rationalized by homology modeling and docking of these hypermodified nucleosides. The modeling suggests that a suitable combination of stabilizing features can partially compensate for the lack of an exocyclic amine, an otherwise important contributor to recognition in the A3AR binding site.