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. The map contains 194 identified proteins, which were detectable by silver stain. All silver stained features were identified by matrix-assisted laser desorption/ionization-time of flight MS of tryptic peptides. Some proteins were found to be represented by multiple features on the 2-D gel. Among the high abundance proteins identified were sets of proteins associated with inflammation, including the classical NFkappaB, p65 (RelA) and NFkappaB, p65 (RelB). We suggest that this composite atlas of the high abundance CF lung epithelial proteome will serve as a reference database for future studies of candidate CF drugs, validating different approaches to CFTR gene therapy, and analogous investigations of other types of human lung disorders.

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. Since no enhancement of A(2B) receptor affinity of 38 was achieved, the previously reported pharmacophore-based searching strategy yielded the most potent and selective structurally-related hit in the database originally searched.

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. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.

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. We used competitive radioligand binding assays to screen flavonoid libraries for affinity and a computational CoMFA analysis of flavonoids to compare steric and electrostatic requirements for ligand recognition at three subtypes of adenosine receptors. Flavone derivatives, such as galangin, were found to bind to three subtypes of adenosine receptors in the microM range. Pentamethylmorin (Ki 2.65 microM) was 14- to 17-fold selective for human A3 receptors than for A1 and A2A receptors. An isoflavone, genistein, was found to bind to A1 receptors. Aurones, such as hispidol (Ki 350 nM) are selective A1 receptor antagonists, and, like genistein, are present in soy. The flavones, chemically optimized for receptor binding, have led to the antagonist, MRS 1067 (3,6-dichloro-2'-(isopropoxy)4'-methylflavone), which is 200-fold more selective for human A3 than A1 receptors. Adenosine receptor antagonism, therefore, may be important in the spectrum of biological activities reported for the flavonoids.

Pyran Template Approach to the Design of Novel A3 Adenosine Receptor Antagonists.

[Table: see text] A3 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 A3 adenosine receptors. These derivatives were synthesized through standard three-component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.e., the central ring served as a molecular scaffold for structurally diverse substituents. We extended this template approach from the DHP series to chemically stable pyran derivatives, in which the ring NH is replaced by O and which is similarly derived from a stepwise reaction of three components. Since the orientation of substituent groups may be conformationally similar to the 1,4-DHPs, a direct comparison between the structure activity relationships of key derivatives in binding to adenosine receptors was carried out. Affinity at human A3 receptors expressed in CHO cells was determined vs. binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-N-methyl-carbamoyladenosine). There was no potency-enhancing effect, as was observed for DHPs, of 4-styryl, 4-phenylethynyl, or 6-phenyl substitutions. The most potent ligands in this group in binding to human A3 receptors were 6-methyl and 6-phenyl analogs, 3a (MRS 1704) and 4a (MRS 1705), respectively, of 3,5-diethyl 2-methyl-4-phenyl-4H-pyran-3,5-dicarboxylate, which had Ki values of 381 and 583 nM, respectively. These two derivatives were selective for human A3 receptors vs. rat brain A1 receptors by 57-fold and 24-fold, respectively. These derivatives were inactive in binding at rat brain A2A receptors, and at recombinant human A2B receptors displayed Ki values of 17.3 and 23.2 µM, respectively. The selectivity, but not affinity, of the pyran derivatives in binding to the A3 receptor subtype was generally enhanced vs. the corresponding DHP derivatives.

Acyl-Hydrazide Derivatives of a Xanthine Carboxylic Congener (XCC) as Selective Antagonists at Human A2B Adenosine Receptors.

The structure-activity relationships (SAR) of 8-phenyl-1,3-dipropylxanthine derivatives in binding to recombinant human A2B 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 p-position of the 8-phenyl substituent [Jacobson et al. (1999): Drug Dev. Res., 47:45-53], a hydrazide and its more stable imide derivatives were synthesized. The hydrazide of XCC (8-[4-[[[carboxy]methyl]oxy]phenyl]-1,3-dipropylxanthine) was acylated with a variety of mono- and dicarboxylic acids. Ki values were determined in the adenosine receptor binding assays. At recombinant human A2B receptors expressed in membranes of HEK-293 cells, antagonist radioligands used were the xanthine 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-oxyacetate-1-propyl-xanthine) and the nonxanthine antagonist [3H]ZM 241385 ([3H]4-(2-[7-amino-2-{furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino-ethyl)phenol). The initial screening utilized rat A1/A2A receptors and human A3 receptors, and selected compounds were examined at the human A1/A2A subtypes. A 1,2-dimethylmaleimide derivative, 14 (MRS 1595), bound to human A2B receptors with a Ki of 19 nM and proved to be selective vs. human A1/A2A/A3 receptors by 160-, 100-, and 35-fold, respectively. Enprofylline (3-propylxanthine) is slightly selective for A2B receptors, suggesting removal of the 1-propyl group; however, combination of the 1-H-3-Pr and 8-phenyl substituents eliminated the selectivity. Other potent and moderately selective A2B antagonists were a tetrahydrophthaloyl derivative 18b (MRS 1614, Ki value 10 nM) and amino acid conjugates of the XCC-hydrazide, i.e., the glutarimide 24b (MRS 1626, Ki value 13 nM), and protected dipeptide 27 (MRS 1615, Ki value 11 nM). Drug Dev. Res. 47:178-188, 1999.

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. Among the 6-phenylazo derivatives, 5'-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y(1) receptors, in guinea-pig vas deferens and bladder P2X(1) receptors, and in ion flux experiments by using recombinant rat P2X(2) receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X(1) receptors in differentiated HL-60 cell membranes was carried out by using [(35)S]ATP-γ-S. A 2'-chloro-5'-sulfo analog of PPADS (C(14)H(12)O(9)N(3)ClPSNa), a vinyl phosphonate derivative (C(15)H(12)O(11)N(3)PS(2)Na(3)), and a naphthylazo derivative (C(18)H(14)O(12)N(3)PS(2)Na(2)), were particularly potent in binding to human P2X(1) receptors. The potencies of phosphate derivatives at P2Y(1) receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C(15)H(13)O(8)N(3)PNa and its m-chloro analog C(15)H(12)O(8)N(3)ClPNa, which were selective for P2X vs. P2Y(1) receptors. C(15)H(12)O(8)N(3)ClPNa was very potent at rat P2X(2) receptors with an IC(50) value of 0.82 µM. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C(14)H(12)-O(8)N(3)ClPSNa) showed high potency at P2Y(1) receptors with an IC(50) of 7.23 µM. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y(1) receptors, whereas at recombinant P2X(2) receptors had an IC(50) value of 1.1 µM. An ethyl phosphonate derivative (C(15)H(15)O(11)N(3)PS(2)Na(3)), whereas inactive at turkey erythrocyte P2Y(1) receptors, was particularly potent at recombinant P2X(2) receptors.

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. Thus, neither A(1)-receptors nor G(i)-proteins appear critically involved in the inhibition of cyclase by riboflavin. Riboflavin did block the stimulation by an adenosine analog of [(35)S]GTPγS binding in rat cerebral cortical membranes. However, riboflavin also inhibited the stimulation by fMLP of [(35)S]GTPγS binding in HL-60 cell membranes. Riboflavin inhibited forskolin-stimulated cyclase in membranes from DDT MF-2 cells > rat adipocytes > PC12 cells, hamster CHO M2 cells, and wild-type S49 cells. There was virtually no inhibition of forskolin-stimulated cyclase in membranes of human platelets, rat cerebral cortex, or cyc(-)S49 cells lacking G(s)-proteins. The calcium-stimulated cyclase in rat cerebral cortical membranes was inhibited by riboflavin. A preincubation of membranes with riboflavin markedly enhanced the inhibition for DDT MF-2 and wild-type and cyc(-)S49 membranes. The extent of inhibition in the different cell lines was dependent on the agent used to stimulate cyclase. Riboflavin, like the P-site inhibitor 2´,5´-dideoxyadenosine, was more potent and efficacious when manganese instead of forskolin was used as the stimulant. However, unlike the P-site inhibitor, riboflavin did not markedly inhibit GppNHp- or fluoride-stimulated cyclase. Riboflavin at low micromolar concentrations appears to have three possibly interrelated effects on second messenger systems subserved by G-proteins. These are antagonism at A(1)-adenosine receptors, inhibition of turnover of guanyl nucleotides at G-proteins, and inhibition of adenylate cyclase.

A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS.

The binding affinities at rat A1, A2a, and A3 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 [3H]PIA or [3H]CGS 21680 in rat brain membranes or [125I]AB-MECA in CHO cells stably transfected with rat A3 receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A3 receptors selectively. Moreover, cytochalasin A, which bound to A1 adenosine receptors with Ki value of 1.9 µM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A1 receptor binding.

Species Differences in Ligand Affinity at Central A3-Adenosine Receptors.

[Table: see text] Binding affinities of purine derivatives at A3 adenosine receptors in different species were compared. Binding was carried out using the novel high affinity agonist ligand [125I]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 A1- and A2a-adenosine antagonist. XAC was added to eliminate binding to non-A3 receptors. In rat brain membranes [125I]AB-MECA exhibited saturable, specific binding with a Kd of 2.28 nM and a Bmax of 43 fmol/mg protein. The affinity of [125I]AB-MECA at the gerbil and rabbit brain A3-receptors was similar to the rat, suggesting that the affinity of this agonist is not highly species dependent. The affinity of various xanthine derivatives was measured in [125I]AB-MECA competition binding assays. Gerbil and rabbit brain A3-receptors were similar in the affinity of antagonists whose potency order in both species was: BWA522 ≥ CPX > XCC, XAC, SPX, BWA1433 > theophylline. The affinities of 8-arylxanthines at the rat, rabbit, and gerbil brain A3 receptors were considerably less than the previously reported affinities at cloned sheep and human A3 receptors. Species differences in agonist affinity were assessed by comparing Ki values at cloned rat brain A3 receptors expressed in CHO cells with cloned sheep and human A3 receptors. Human and rat brain A3 receptors were highly similar in the relative affinities of agonists, and sheep brain A3 receptors were unlike either human or rat A3 receptors in agonist affinity.

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. The irreversibility is likely due to the presence of the chemically reactive isothiocyanate group, since the binding of the corresponding analogue in which the isothiocyanate was replaced with a chloro group was completely reversible. The potency of ISC to irreversibly inhibit the binding of [(3)H]CGS 21680 in several species varied in the order rat ≈ guinea pig > bovine ≈ rabbit. In all four species, binding of the A(1)-selective agonist [(3)H]R-N(6)-phenylisopropyladenosine was not diminished by pre-treatment with 2 µM ISC. The kinetics of irreversible inhibition of rat A(2a)-receptors by 2 µM ISC gave a t(1/2) of approximately 3 min. Following partial inactivation, the remaining rat A(2a)-binding sites retained the same K(d) value as in control membranes for saturation by [(3)H]CGS 21680. Thus, ISC appears to be a selective affinity label for A(2a)- versus A(1)-receptors in the brain.

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. Selective chemical affinity labels for A(1) and A(2a) receptors have been introduced. For example, an isothiocyanate, p-DITC-APEC (100 nM), irreversibly diminished the B(max) for [(3)H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5'-N-ethylcarboxamidoadenosine) binding in rabbit striatal membranes by 71% (K(d) unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A(2a) receptors were characterized using [(3)H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-l,3-dipropylxanthine), respectively. The inhibition of A(2) radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A(1) receptors in which either His(251) or His(278) residues have been substituted with Leu suggest that both histidines are important in binding.