MRS 1220

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism.[Pubmed:24970394]

FASEB J. 2014 Oct;28(10):4211-22.

In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, tauD2 and tauD3, that diffused at 2.29 +/- 0.35 and 0.09 +/- 0.03 mum(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 mum(2)/s). The binding of CA200645 to tauD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting tauD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with tauD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface

A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve.[Pubmed:24311446]

Glia. 2014 Feb;62(2):199-216.

Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N -methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.

Roles of adenosine receptor subtypes on the antinociceptive effect of sildenafil in rat spinal cord.[Pubmed:20547208]

Neurosci Lett. 2010 Aug 23;480(3):182-5.

We recently found that the antinociceptive effects produced by intrathecal administration of sildenafil, a phosphodiesterase 5 inhibitor, were reversed by a nonspecific adenosine receptor antagonist, suggesting that adenosine receptors are involved in sildenafil-induced antinociception. Four adenosine receptor subtypes have been identified: A(1), A(2A), A(2B), and A(3). We examined the involvement of spinal adenosine receptor subtypes in the antinociceptive effects of intrathecal sildenafil. Intrathecal catheters were implanted in male SD rats, and nociception was assessed using the formalin test, which consisted of a subcutaneous injection of 50 microl of 5% formalin solution into the hind paw. We examined the effects of an adenosine A(1) receptor antagonist (CPT), an adenosine A(2A) receptor antagonist (CSC), an adenosine A(2B) receptor antagonist (alloxazine), and an adenosine A(3) receptor antagonist (MRS 1220) on sildenafil-induced antinociception. Intrathecal sildenafil suppressed formalin-induced flinching during phases 1 and 2 of the test in a dose-dependent manner. Intrathecal CPT, CSC, alloxazine, and MRS 1220 all suppressed the antinociceptive effects of sildenafil during both phases of the formalin test. These results suggest that sildenafil is an effective treatment for acute pain and the facilitated pain state at the spinal level. Additionally, spinal adenosine A(1), A(2A), A(2B), and A(3) receptors may play a role in sildenafil-induced antinociception.

The role of adenosine receptors in regulating production of tumour necrosis factor-alpha and chemokines by human lung macrophages.[Pubmed:20136829]

Br J Pharmacol. 2010 Mar;159(6):1304-11.

BACKGROUND AND PURPOSE: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines. EXPERIMENTAL APPROACH: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. KEY RESULTS: LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors. CONCLUSIONS AND IMPLICATIONS: LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.

Adenosine blocks IFN-gamma-induced phosphorylation of STAT1 on serine 727 to reduce macrophage activation.[Pubmed:19846878]

J Immunol. 2009 Nov 15;183(10):6767-77.

Macrophages are activated by IFN-gamma, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-gamma signaling induces phosphorylation of two STAT1 residues: Tyr(701) (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser(727) (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-gamma signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-gamma-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-gamma-induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A(3) receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25-50%. Further, RAW 264.7 A(3) receptor stimulation with Cl-IB-MECA reduces IFN-gamma-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A(3) receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-gamma-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-gamma-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.

Adenosine A1 and A3 receptor agonists inhibit nonadrenergic, noncholinergic relaxations in the guinea pig isolated trachea.[Pubmed:19077382]

Respiration. 2009;78(1):75-83.

BACKGROUND: Adenosine affects the tone and reactivity of airways by activating specific membrane receptors, named A(1), A(2a), A(2b) and A(3). It affects cellular activities either directly by regulating membrane ion exchanges and polarization, or indirectly by modifying neurotransmitter release. OBJECTIVES: We assessed the effect of A(1) and A(3) receptor activation on electrically induced nonadrenergic, noncholinergic (NANC) relaxations in the guinea pig isolated trachea and the localization of A(1) and A(3) receptors in tracheal inhibitory neurons. METHODS: NANC responses at 3 Hz were evaluated in the presence of 2-chloro-N(6)-cyclopentyladenosine (CCPA), a selective A(1) agonist, and 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective A(3) agonist, before and after the administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) antagonist, or 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino[1,2,4]triazolo[1,5-c])quinazoline (MRS 1220), a selective A(3) antagonist, respectively. For immunohistochemistry, tissues were exposed to antibodies to HuC/D, a general neuronal marker, neuronal nitric oxide synthase (nNOS), and A(1) or A(3) adenosine receptors and processed by indirect immunofluorescence. RESULTS: CCPA (10 nM-3 microM) inhibited NANC relaxations. DPCPX (10 nM) failed to antagonize the effect of CCPA, but inhibited per se NANC relaxations (range 0.1-100 nM). CCPA (10 nM-10 microM) contracted unstimulated tracheal preparations, an effect antagonized by 10 nM DPCPX, with a pK(B) value of 8.43. Cl-IB-MECA (10 nM-3 microM) inhibited NANC relaxations through a mechanism antagonized by MRS 1220 (100 nM). A(1)- and A(3)-positive neurons containing nNOS were detected in tracheal sections. CONCLUSIONS: Enogenous adenosine may induce airway hyperresponsiveness by inhibiting NANC relaxations via A(1) and A(3) receptors.

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes.[Pubmed:9667972]

J Med Chem. 1998 Jul 16;41(15):2835-45.

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.

Pharmacological characterization of novel A3 adenosine receptor-selective antagonists.[Pubmed:9364471]

Neuropharmacology. 1997 Sep;36(9):1157-65.

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 triazoloquinazoline 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-gamma-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-alpha formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 microM.

Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype.[Pubmed:8863790]

J Med Chem. 1996 Oct 11;39(21):4142-8.

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) binds to human A3 receptors with high affinity (Ki = 14 nM), while it lacks affinity at rat A3 receptors. Acylated derivatives of the 5-amino group and other modifications were prepared in an effort to provide A3 subtype selectivity. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA ([3H]-(R)-N6-(phenylisopropyl)-adenosine) and [3H]CGS 21680 ([3H]-2-[[4-(2-carboxy ethyl)phenyl]ethylaminol]-5'-(N- ethyl- carbamoyl)adenosine), respectively. Affinity was determined at cloned human A3 receptors using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine). A series of straight chain alkyl amides demonstrated that the optimal chain length occurs with the 5-N-propionyl derivative, 3, which had a Ki value of 7.7 nM at human A3 receptors, and was 40- and 14-fold selective vs rat A1 and A2A receptors, respectively. The 5-N-benzoyl derivative, 10, displayed Ki values of 680 and 273 nM at rat A1 and A2A receptors, respectively, and 3.0 nM at human A3 receptors. A 5-N-phenylacetyl derivative, 12, was 470-fold selective for human A3 vs rat A1 receptors with a Ki value of 0.65 nM. A conjugate of Boc-gamma-aminobutyric acid was also prepared but was nonselective. Conversion of the 5-amino group of CGS15943 to an oxo function resulted in lower affinity but 15-fold selectivity for human A3 receptors.