Probing the adenosine receptor with adenosine and xanthine biotin conjugates

Biotin-containing analogs of a potent agonist (N6-phenyladenosine) and a potent antagonist (1,3-dipropyl-8-phenylxanthine) of adenosine receptor activity have been synthesized. A spacer chain to the biotin moiety is attached in both cases to the para-position of the phenyl ring. Two biotin conjugates of N6-phenyladenosine differing only in the length of the spacer chain bind to the adenosine receptor and to avidin simultaneously. The shorter-chain derivative was more potent in inhibiting binding of N6-[3H]cyclohexyladenosine to rat cerebral cortical membranes (Ki of 11 nM in the absence of avidin, 36 nM for the avidin complex). Three biotin conjugates of 1,3-dipropyl-8-phenylxanthine bound competitively to the adenosine receptor, but only in the absence of avidin. The results are interpreted in terms of the possible orientation of the ligands at the receptor binding site.     

Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine

Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5?and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.     

GPCR ligand dendrimer (GLiDe) conjugates: adenosine receptor interactions of a series of multivalent xanthine antagonists

Previously, G protein-coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here, we prepared GPCR ligand--dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson's disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach K(i) values in the nanomolar range. The order of affinity of each conjugate was hA(2A)AR > hA(3)AR > hA(1)AR, while the corresponding monomer was ranked hA(2A)AR > hA(1)AR ≥ hA(3)AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the G(i)-coupled A(1)AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration--response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its K(i) value), it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading.     

PD 116,948, a highly selective A1 adenosine receptor antagonist

(R)-N-(1-Methyl-2-phenylethyl) adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic activity and coronary vasodilator activity. These actions of R-PIA are proposed to be mediated by subtypes (A1 and A2) of adenosine receptors. PD 116,948 is a xanthine derivative which is a highly selective A1 adenosine receptor ligand. In this study PD 116,948 selectively antagonized the negative chronotropic activity of R-PIA in the isolated rat heart. These results are consistent with, and add further support to the hypothesis that adenosine receptor agonists mediate their negative chronotropic activity via A1 receptors and their vasodilator activity via A2 receptors.     

The A1 adenosine receptor antagonist 1,3, dipropyl-8-cyclopentylxanthine (DPCPX) displays adenosine agonist properties in the FRTL5 thyroid cell line

We have evaluated whether the type I adenosine receptor mediates adenosine's ability to inhibit thyrotropin-stimulated cyclic AMP generation and DNA synthesis in FRTL5 cells. The xanthine derivative 1,3-dipropyl-8-cyclopentylxanthine, a selective antagonist for the type 1 adenosine receptor, binds to FRTL5 with high affinity and specificity. 1,3-Dipropyl-8-cyclopentylxanthine does not alter basal cyclic AMP levels but does reverse adenosine's ability to inhibit thyrotropin-stimulated cyclic AMP generation. 1,3-Dipropyl-8-cyclopentylxanthine also potently inhibits thyrotropin-stimulated and dibutyryl cyclic AMP-stimulated [3H]-thymidine incorporation into DNA in FRTL5 cells. Thus, in FRTL5 cells, 1,3-dipropyl-8-cyclopentylxanthine displays both adenosine antagonist and adenosine agonist properties, the latter occurring at a site distal to cyclic AMP generation.     

Effects of pentobarbital tolerance and dependence on convulsant and GABAA receptor antagonist binding.

Experiments were performed which examined the effects of pentobarbital tolerance and dependence on GABAA receptor antagonist binding. In rats implanted with pentobarbital pellets for 7 days, followed by 24 hours of withdrawal, there was a significant decrease in the latency of TBPS-induced seizures and an increase in [35S]TBPS binding in the frontal cortex. The pentobarbital tolerant rats had a significant increase in the low affinity KD of [3H]SR95531 binding. Removal of the pellets for 24 hours caused a reversal of the effect on the low affinity KD and caused a decrease in the number of low affinity binding sites. In vitro addition of pentobarbital to binding assays produced a decrease in the number of high affinity [3H]SR95531 binding sites without changing low affinity binding. In the cerebellum, the binding in none of the treatment groups was significantly different from placebo. These observations suggest that pentobarbital tolerance and withdrawal cause changes in the properties of the GABAA receptor antagonist binding site which are different from those caused by in vitro exposure to the drug.     

The discovery of a selective, high affinity A(2B) adenosine receptor antagonist for the potential treatment of asthma

Adenosine has been suggested to play a role in asthma, possibly via activation of A(2B) adenosine receptors on mast cells and other pulmonary cells. We describe our initial efforts to discover a xanthine based selective A(2B) AdoR antagonist that resulted in the discovery of CVT-5440, a high affinity A(2B) AdoR antagonist with good selectivity (A(2B) AdoR K(i)=50 nM, selectivity A(1)>200: A(2A)>200: A(3)>167).     

Inhibitory effect of KW-3902, an adenosine A(1) receptor antagonist, on p-aminohippurate transport in OK cells.

KW-3902 (8-(noradamantan-3-yl)-1,3-dipropylxanthine) is a novel potent and selective adenosine A(1) receptor antagonist. We examined the effect of KW-3902 on p-aminohippurate (PAH) transport in opossum kidney (OK) epithelial cells. Pretreatment for 3 h with KW-3902 inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side. The uptake of PAH across the basolateral membrane of OK cells was inhibited by KW-3902 pretreatment in a time- and concentration-dependent manner. A kinetic analysis revealed that the inhibitory effect of KW-3902 on the basolateral PAH uptake was due to an increase in the Michaelis constant (K(m)) as well as a decrease in the maximum uptake rate (V(max)), showing that the inhibition was a mixed type. Pretreatment with adenosine deaminase or 8-cyclopentyl-1,3-dipropylxanthine, another selective adenosine A(1) receptor antagonist, also decreased the basolateral PAH uptake. KW-3902 pretreatment had no effect on the concentration of intracellular alpha-ketoglutarate which exchanges for PAH across the basolateral membrane of OK cells. These results suggest that KW-3902 has an inhibitory effect on PAH transport in OK epithelial cells.     

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice

Introduction  

Endothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice.     

Chemiluminescent determination of adenosine, inosine, and hypoxanthine/xanthine

A fully automatic method for analysis of adenosine, inosine, and hypoxanthine/xanthine which combines the specificity of enzymatic catalysis and sensitivity of chemiluminescence is presented. The hydrogen peroxide formed by sequential catabolism of purines to uric acid is detected by the oxidation of luminol in the presence of peroxidase. The method takes advantage of the fact that light output in the H2O2/luminol system is transient. By adopting a two-step procedure this feature enables selective determination of adenosine, inosine, and hypoxanthine/xanthine. In step 1 any purines lower in the catabolic sequence than the analyte under study are converted to uric acid. Light emission is allowed to decay to baseline levels. During step 2 the analyte is selectively degraded. The H2O2 formed leads to a new light emission which is proportional to the square of analyte concentration. The method can be performed with commercially available reagents and enzymes and requires minimal processing of biological samples. Excellent agreement has been obtained with HPLC analysis. Sensitivity is in the range of 5-10 nmol/liter in as little as 0.1 ml. More than 200 samples per day can be analyzed by a single operator.     

Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor

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 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 an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.     

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro3)GIP receptor antagonist in type 2 diabetes

In this study, we tested the biological activity of a novel acylated form of (Pro3)glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys16 to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro3)GIP, (Pro3)GIPLys16PAL was completely stable to the actions of DPP-IV and significantly (p<0.01 to p<0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro3)GIPLys16PAL also significantly (p<0.05 to p<0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro3)GIPLys16PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro3)GIPLys16PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p<0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p<0.05) and pancreatic insulin was significantly reduced (p<0.05) in the (Pro3)GIPLys16PAL-treated mice. These data demonstrate that acylation of Lys16 with palmitic acid in (Pro3)GIP does not improve its biological effectiveness as a GIP receptor antagonist.     

Design and evaluation of xanthine based adenosine receptor antagonists: Potential hypoxia targeted immunotherapies

Molecular modeling techniques were applied to the design, synthesis and optimization of a new series of xanthine based adenosine A_2A receptor antagonists. The optimized lead compound was converted to a PEG derivative and a functional in vitro bioassay used to confirm efficacy. Additionally, the PEGylated version showed enhanced aqueous solubility and was inert to photoisomerization, a known limitation of existing antagonists of this class.     

Aryl hydrocarbon receptor (AhR)-mediated induction of xanthine oxidase/xanthine dehydrogenase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, induced xanthine oxidase and xanthine dehydrogenase (XO/XDH) activities, in addition to ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in liver of mice. When TCDD was given to mice as a single oral dose of 40 microg/kg, the activities of XO and XDH increased about threefold within 3 days and the increased levels were maintained for 4 weeks. The treatment of mice with 3-methylcholanthrene also induced XO/XDH activities, but phenobarbital and dexamethasone had no effect. The level of aldehyde oxidase, a molybdenum flavoenzyme related to XO/XDH, in mouse liver was also enhanced about 1.5-fold by TCDD treatment. The inducing effect of TCDD and 3-methylcholanthrene was not observed in null mice (AhR(-/-)), which lack the AhR gene. XO and XDH activities were induced by TCDD in heterozygous mice (AhR(+/-)). The lipid peroxidation in liver was stimulated by TCDD. The induction of XO and XDH, which produces reactive oxygen species, may contribute to the various toxicities of TCDD.     

Potent S1P receptor agonists replicate the pharmacologic actions of the novel immune modulator FTY720

Alteration in lymphocyte trafficking and prevention of graft rejection in rodents observed on exposure to FTY720 (1) or its corresponding phosphate ester 2 can be induced by the systemic administration of potent sphingosine-1-phosphate receptor agonists exemplified by 19. The similar S1P receptor profiles of 2 and 19 coupled with their comparable potency in vivo supports a connection between S1P receptor agonism and immunosuppressive efficacy.     

CGS 15943, An adenosine A2 receptor antagonist,reduces cerebral ischemic injury in the mongolian gerbil

The adenosine A₂ receptor antagonist CGS 15943 (0.1 mg/kg, i.p.) was tested for cerebroprotective activity in a gerbil stroke model. CGS 15943 markedly reduced stroke injury assessed by locomotor activity monitoring and by histopathological measurement of hippocampal CA1 pyramidal cell injury. It is proposed that a previously demonstrated reduction in the ischemia/reperfusion-evoked release of excitotoxic amino acids following CGS 15943 administration could account for its cerebroprotective actions.     

Potent benzimidazolone based human beta(3)-adrenergic receptor agonists

The synthesis and biological evaluation of a series of benzimidazolone beta(3) adrenergic receptor agonists are described. A trend toward the reduction of rat atrial tachycardia upon increasing steric bulk at the 3-position of the benzimidazolone moiety was observed.