High abundance protein profiling of cystic fibrosis lung epithelial cells.

Protein profiles of cultured cystic fibrosis (CF) lung epithelial cells were analyzed by two-dimensional gel electrophoresis and mass spectrometry (MS). The analysis gave rise to a protein map over the pI range of 4-7, and a molecular weight range of ca. 100-10 kDa.

Quinazolines as adenosine receptor antagonists: SAR and selectivity for A2B receptors.

We have recently reported the discovery of numerous new compounds that are selective inhibitors of all of the subtypes of the adenosine receptor family via a pharmacophore database searching and screening strategy. During the course of this work we made the unexpected discovery of a potent A(2B) receptor antagonist, 4-methyl-7-methoxyquinazolyl-2-(2'-amino-4'-imidazolinone) (38, CMB 6446), which showed selectivity for this receptor and functioned as an antagonist, with a binding K(i) value of 112 nM. We explored the effects of both substituent- and ring-structural variations on the receptor affinity in this series of derivatives, which were found to be mostly non-selective adenosine receptor ligands with K(i) values in the micromolar range.

Quantitation of the P2Y(1) receptor with a high affinity radiolabeled antagonist.

2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors.

Interactions of flavones and other phytochemicals with adenosine receptors.

Dietary flavonoids have varied effects on animal cells, such as inhibition of platelet binding and aggregation, inhibition of inflammation, and anticancer properties, but the mechanisms of these effects remain largely unexplained. Adenosine receptors are involved in the homeostasis of the immune, cardiovascular, and central nervous systems, and adenosine agonists/antagonists exert many similar effects. The affinity of flavonoids and other phytochemicals to adenosine receptors suggests that a wide range of natural substances in the diet may potentially block the effects of endogenous adenosine.

Pyran Template Approach to the Design of Novel A Adenosine Receptor Antagonists.

[Table: see text] A adenosine receptor antagonists have potential as anti-inflammatory, anti-asthmatic, and anti-ischemic agents. We previously reported the preparation of chemical libraries of 1,4-dihydropyridine (DHP) and pyridine derivatives and identification of members having high affinity at A adenosine receptors. These derivatives were synthesized through standard three-component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.

Acyl-Hydrazide Derivatives of a Xanthine Carboxylic Congener (XCC) as Selective Antagonists at Human A Adenosine Receptors.

The structure-activity relationships (SAR) of 8-phenyl-1,3-dipropylxanthine derivatives in binding to recombinant human A adenosine receptors were explored, in order to identify selective antagonists. Based on the finding of receptor selectivity in MRS 1204, containing an N-hydroxysuccinimide ester attached through the -position of the 8-phenyl substituent [Jacobson et al. (1999): Drug Dev.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal-5'-phosphate-6-phenylazo-2',4'-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated.

Riboflavin: Inhibitory Effects on Receptors, G-Proteins, and Adenylate Cyclase.

Riboflavin inhibited binding of both agonist and antagonist radioligands to rat brain A(1)-adenosine receptors with K(i) values of approximately 10 µM. In an adenylate cyclase assay with membrane preparations from either rat adipocytes or DDT MF-2 cells, both of which contain A(1)-adenosine receptors, riboflavin inhibited isoproterenol-stimulated cyclase activity with an IC(50) of approximately 20 µM. However, the inhibition of cyclase by riboflavin was not reversed by an A(1)-selective antagonist, nor by pretreatment with pertussis toxin.

A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS.

The binding affinities at rat A, A, and A adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [H]PIA or [H]CGS 21680 in rat brain membranes or [I]AB-MECA in CHO cells stably transfected with rat A receptors. Several new classes of adenosine antagonists ( 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified.

Species Differences in Ligand Affinity at Central A-Adenosine Receptors.

[Table: see text] Binding affinities of purine derivatives at A adenosine receptors in different species were compared. Binding was carried out using the novel high affinity agonist ligand [I]AB-MECA (3-iodo-4-aminobenzyladenosine-5'-N-methyluronamide) in the presence of 1.0 μM XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine), an A- and A-adenosine antagonist.

8-(3-Isothiocyanatostyryl)caffeine Is a Selective, Irreversible Inhibitor of Striatal A(2)-Adenosine Receptors.

8-(3-Isothiocyanatostyryl)caffeine (ISC) was synthesized and shown to inhibit selectively the binding of [(3)H]CGS 21680 (an A(2a)-selective agonist) at adenosine receptors in striatal membranes. The K(i) value at A(2a)-receptors was found to be 110 nM (rat), with selectivity ratios for A(2a) versus A(1)-receptors in rat, guinea pig, bovine, and rabbit striatum of >100-fold. Preincubation of membranes with ISC caused a dose-dependent, irreversible antagonism of the binding of [(3)H]CGS 21680, with an IC(50) value of 3 μM.

Molecular Characterization of A(1) and A(2a) Adenosine Receptors.

Detailed amino acid sequence analyses of A(1) and A(2a) 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 A(2a) receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC.