Cellular immune response to human cartilage glycoprotein-39 (HC gp-39)-derived peptides in rheumatoid arthritis and other inflammatory conditions.

OBJECTIVE: To study the specificity of the peripheral blood mononuclear cell (PBMC) response to peptides derived from human cartilage glycoprotein-39 (HC gp-39) in patients with rheumatoid arthritis (RA) and the correlation between this response and disease activity. METHODS: RA patients, patients with systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) or osteoarthritis (OA) and healthy controls were studied. All individuals were typed for HLA-DRB1 and their disease activity score was documented. Proliferation of PBMC was measured following incubation with five different HC gp-39-derived peptides, selected by the use of a DR4 (DRB1*0401) binding motif. RESULTS: A proliferative response to one of the five peptides (peptide 259-271 at 10 microg/ml) was more often observed in RA patients than in healthy controls (P=0.001). RA patients who expressed DRB1*0401 more often showed a response against this peptide than RA patients who did not express this RA-associated haplotype. This response was not RA-specific since patients with IBD or OA also showed a response significantly more frequently than healthy controls (P:=0.02 and P=0.03 respectively). However, the level of the response against peptide 259-271 correlated with disease activity in RA patients but not in patients with IBD or SLE. Increased responses to HC gp-39 263-275 were found in patients with IBD or OA; a trend towards such a response failed to reach significance in RA patients in this study. CONCLUSION: In RA patients as well as in patients with other inflammatory conditions, HC gp-39-derived peptides may be targets of the T-cell-mediated immune response. In the RA patient group the immune response to HC gp-39-derived peptide 259-271 correlated with disease activity.

1-Aminoisoquinoline as benzamidine isoster in the design and synthesis of orally active thrombin inhibitors.

Replacement of the highly basic benzamidine moiety of NAPAP by the moderately basic 1-aminoisoquinoline moiety resulted in thrombin inhibitors with improved selectivity towards trypsin and enhanced Caco-2 cell permeability.

Modelling the P2Y purinoceptor using rhodopsin as template.

The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).

Correlation of binding affinities with non-bonded interaction energies of thrombin-inhibitor complexes.

Several empirical modeling protocols are evaluated allowing a quantification of the interaction between an enzyme and a series of inhibitors. The evaluation and optimization of the modeling protocols used a database of 35 non-covalently bound inhibitors of human thrombin. Intermolecular interaction energies were calculated with CHARMm after energy minimization of the modeled complexes using various dielectric functions and constraining strategies. These calculated binding energies were correlated with the experimentally obtained binding constants of the inhibitors. The best protocols resulted in linear correlations with correlation coefficients > 0.80. In the best protocols the enzyme was fully constrained, the ligand was allowed to move freely and electrostatics were scaled down drastically or fully neglected during the energy minimization. For the interaction energy evaluation step, a distance-dependent dielectric function epsilon = R proved to be optimal. This simple empirical protocol, that neglects solvation or entropy effects, can be implemented readily in other force field packages and may be applied on various enzyme-inhibitor complexes, providing a tool for the evaluation and rank-ordering of newly designed inhibitors.

Stimulation by alkylxanthines of chloride efflux in CFPAC-1 cells does not involve A1 adenosine receptors.

A series of 8-substituted derivatives of 1,3,7-alkylxanthines was synthesized as potential activators of chloride efflux from a human epithelial cell line (CFPAC) expressing the cystic fibrosis transmembrane regulator (CFTR) delta F508 mutation. Their interactions with rat brain A1 and A2a receptors were also studied in radioligand binding experiments. Substitution was varied at the xanthine 1-, 3-, 7- and 8-positions. 1,3-Dipropyl-8-cyclopentylxanthine (CPX) stimulated Cl- efflux in the 10(-8) M range, with a maximal effect reaching 200% of control and diminishing at higher concentrations. The potent adenosine antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3-dipropylxanthine, nonselective at human A1 and A2a receptors, was inactive in Cl- efflux. 1,3-Diallyl-8-cyclohexylxanthine (DAX) was highly efficacious in stimulating chloride efflux with levels reaching > 300% of control, although micromolar concentrations were required. 1,3,7-Trimethyl-8-(3-chlorostyryl)xanthine, an A2a-selective adenosine antagonist, was only weakly active. Caffeine, which acts as an nonselective adenosine antagonist in the range of 10(-5) M, was active in Cl- efflux in the low nanomolar range but with low efficacy. Thus, among the xanthine derivatives of diverse structure, there was no correlation between potency in Cl- efflux and adenosine antagonism. Poly(A)+ RNA isolated from CFPAC-1 cells showed no hybridization to a human A1 receptor cDNA probe, excluding this receptor as a mediator of CPX-elicited Cl- efflux. Thus, this action of xanthines in stimulating Cl- efflux in CFPAC cells, which express a defective CFTR, represents a novel site of action apparently unrelated to adenosine receptors.

A binding site model and structure-activity relationships for the rat A3 adenosine receptor.

A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a receptors, substitutions at the N6 and 5' positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5'-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5'-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5'-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 microM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.

Molecular modeling of adenosine receptors. The ligand binding site on the rat adenosine A2A receptor.

The amino acid sequence of the rat adenosine A2A receptor and the atomic coordinates of bacteriorhodopsin were combined to generate a three-dimensional model for the adenosine A2A receptor. This model consists of seven amphipathic alpha-helices, forming a pore that is rather hydrophilic compared to the hydrophobic outside of the protein. Subsequently, a highly potent and selective ligand for this receptor, 2-(cyclohexylmethylidinehydrazino)adenosine (SHA 174), was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Subsequently, the deduced binding site was used to model other potent ligands, including 8-(3-chlorostyryl)caffeine, a new A2-selective antagonist, that could all be accommodated consistent with earlier biochemical and pharmacological findings. Finally, some thoughts on how adenosine receptor activation might proceed are put forward, based on structural analogies with the enzyme family of serine proteases.

Structure-activity relationships of N6-benzyladenosine-5'-uronamides as A3-selective adenosine agonists.

Adenosine analogues modified at the 5'-position as uronamides and/or as N6-benzyl derivatives were synthesized. These derivatives were examined for affinity in radioligand binding assays at the newly discovered rat brain A3 adenosine receptor and at rat brain A1 and A2a receptors. 5'-Uronamide substituents favored A3 selectivity in the order N-methyl > N-ethyl approximately unsubstituted carboxamide > N-cyclopropyl. 5'-(N-Methylcarboxamido)-N6-benzyladenosine was 37-56-fold more selective for A3 receptors. Potency at A3 receptors was enhanced upon substitution of the benzyl substituent with nitro and other groups. 5'-N-Methyluronamides and N6-(3-substituted-benzyl)adenosines are optimal for potency and selectivity at A3 receptors. A series of 3-(halobenzyl)-5'-N-ethyluronamide derivatives showed the order of potency at A1 and A2a receptors of I approximately Br > Cl > F. At A3 receptors the 3-F derivative was weaker than the other halo derivatives. 5'-N-Methyl-N6-(3-iodobenzyl)adenosine displayed a Ki value of 1.1 nM at A3 receptors and selectivity versus A1 and A2a receptors of 50-fold. A series of methoxybenzyl derivatives showed that a 4-methoxy group best favored A3 selectivity. A 4-sulfobenzyl derivative was a specific ligand at A3 receptors of moderate potency. An aryl amino derivative was prepared as a probe for radioiodination and receptor cross-linking.

The N-terminal coiled-coil domain of beta is essential for gamma association: a model for G-protein beta gamma subunit interaction.

We have identified the N terminus of the beta subunit as an essential domain for G-protein beta gamma assembly. A C-terminal fragment, beta 1-(130-340), fails to bind gamma unless coexpressed with the complementary N-terminal fragment, beta 1-(1-129). Deletion of the N-terminal 33 residues of beta 1, a region identified by computer algorithm to favor coiled-coil formation, abolishes gamma 2 association. On the basis of these findings, we propose a coiled-coil model of beta gamma interaction and refine this by computer-assisted molecular modeling. The model is tested by further mutagenesis: reversing the charge of residues in beta 1 that are hypothesized to be involved in interhelical salt bridges precludes gamma association. Insertions in the coiled-coil region, which disrupt the proposed hydrophobic interface, prevent gamma association. This structural basis for beta gamma dimerization provides a starting point for the design of beta and gamma mutants that can be used to map regions in beta gamma critical for interactions with the alpha subunit, receptors, and effectors.

Structure-activity relationships of 8-styrylxanthines as A2-selective adenosine antagonists.

A series of substituted 8-styryl derivatives of 1,3,7-alkylxanthines was synthesized as potential A2-selective adenosine receptor antagonists, and the potency at rat brain A1- and A2-receptors was studied in radioligand binding experiments. At the xanthine 7-position, only small hydrophobic substituents were tolerated in receptor binding. 7-Methyl analogues were roughly 1 order of magnitude more selective for A2 versus A1 receptors than the corresponding 7-H analogues. 1,3-Dimethylxanthine derivatives tended to be more selective for A2-receptors than the corresponding 1,3-diallyl, diethyl, or dipropyl derivatives. Substitutions of the phenyl ring at the 3-(monosubstituted) and 3,5-(disubstituted) positions were favored. 1,3, 7-Trimethyl-8-(3-chlorostyryl)xanthine was a moderately potent (Ki vs [3H]CGS 21680 was 54 nM) and highly A2-selective (520-fold) adenosine antagonist. 1,3,7-Trimethyl-8-[(3-carboxy-1-oxopropyl)amino] styryl]xanthine was highly A2-selective (250-fold) and of enhanced water solubility (max 19 mM). 1,3-Dipropyl-7-methyl-8-(3,5-dimethoxystyryl) xanthine was a potent (Ki = 24 nM) and very A2-selective (110-fold) adenosine antagonist.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL 2-THIO DERIVATIVES OF ATP.

2-Alkylthio analogues of adenosine 5'-triphosphate were synthesized and evaluated as P2y purinoceptor agonists. ATP and analogues transiently increased intracellular Ca2+ levels in C6 glioma cells and in skeletal muscle derived myotubes in culture. Most derivatives were resistant to stepwise dephosphorylation by ecto-ATPases.

Synthesis and biological activity of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine: water-soluble and peripherally selective adenosine agonists.

A series of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine was synthesized, revealing that N6-(p-sulfophenyl)adenosine (10b) is a moderately potent (Ki vs [3H]PIA in rat cortical membranes was 74nM) and A1-selective (120-fold) adenosine agonist, of exceptional aqueous solubility of > 1.5 g/mL (approximately 3 M). Compound 10b was very potent in inhibiting synaptic potentials in gerbil hippocampal slices with an IC50 of 63 nM. At a dose of 0.1 mg/kg ip in rats, 10b inhibited lipolysis (a peripheral A1 effect) by 85% after 1 h. This in vivo effect was reversed using the peripherally selective A1-antagonist 1,3-dipropyl-8-[p-(carboxyethynyl)phenyl]xanthine (BW1433). The same dose of 10b in NIH Swiss mice (ip) was nearly inactive in locomotor depression, an effect that has been shown to be centrally mediated when elicited by lower doses of other potent adenosine agonists, such as N6-cyclohexyladenosine (CHA) (Nikodijevic et al. FEBS Lett. 1990, 261, 67). HPLC studies of biodistribution of a closely related and less potent homologue, N6-[4-(p-sulfophenyl)butyl]adenosine indicated that a 25 mg/kg ip dose in mice resulted in a plasma concentration after 30 min of 0.46 micrograms/mL and no detectable drug in the brain (detection limit < 0.1% of plasma level). Although 10b at doses > 0.1 mg/kg in mice depressed locomotor activity, this depression was unlike the effects of CHA and was reversible by BW1433. These data suggest that 10b is a potent adenosine agonist in vivo and shows poor CNS penetration.

A1 adenosine-receptor antagonists activate chloride efflux from cystic fibrosis cells.

A1 adenosine-receptor-antagonist drugs such as 8-cyclopentyl-1,3-dipropylxanthine (CPX) and xanthine amine congener (XAC) are found to activate the efflux of 36Cl- from CFPAC cells. These cells are a pancreatic adenocarcinoma cell line derived from a cystic fibrosis (CF) patient homozygous for the common mutation, deletion of Phe-508. The active concentrations for these compounds are in the low nanomolar range, consistent with action on A1 adenosine receptors. In addition, drug action can be blocked by exogenous agonists such as 2-chloroadenosine and also can be antagonized by removal of endogenous agonists by treatment with adenosine deaminase. Cells lacking the CF genotype and phenotype, such as HT-29 and T84 colon carcinoma cell lines, appear to be resistant to activation of chloride efflux by either drug. CFPAC cells transfected with the CF transmembrane regulator gene, CFTR, are also resistant to activation by CPX. We conclude that, since these antagonists are of relatively low toxicity and appear to act somewhat selectively, they might be considered as promising therapeutic candidates for CF.

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

Molecular modeling of adenosine receptors. I. The ligand binding site on the A1 receptor.

The amino acid sequence of the canine adenosine A1 receptor and the atomic coordinates of a structurally related protein, bacteriorhodopsin, were combined to generate a three-dimensional model for the adenosine A1 receptor. This model consists of seven amphipathic alpha-helices, forming a pore that has a rather distinct partition between hydrophobic and hydrophilic regions. Subsequently, a highly potent and selective ligand, N6-cyclopentyladenosine, was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand, obtained from indirect modeling studies by the 'active analog approach'. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Finally, the deduced binding site was used to model other potent ligands that could all be accommodated consistent with earlier biochemical and pharmacological findings.

Mapping the xanthine C8-region of the adenosine A1 receptor with computer graphics.

Substitution at the 8-position of 1,3-dipropylxanthines can lead to very potent and selective adenosine A1 antagonists. The xanthine C8-region was investigated in this study, using CAMM (computer-assisted molecular modeling). This region can be divided into two subregions with a considerable overlap in volume: a phenyl region which binds the flat substituents and a cycloalkyl region which binds the other substituents. The 8-phenyl-substituted derivatives bind with an N9-C8-Cl'-C2' dihedral angle of 220 degrees; this dihedral angle is 330 degrees for the 8-cycloalkyl-substituted derivatives. The lower affinity of C8-substituted 7-methyl-1,3-dipropylxanthines can be explained quantitatively with steric hindrance, which C8-substituents experience from the 7-methyl group in these conformations. The substitution pattern determines the affinity for 8-phenyl-substituted compounds for which the energy cost to reach the dihedral angle of 220 degrees is low, but has little influence otherwise. The affinity of the 8-cycloalkyl-1,3-dipropylxanthines is mainly volume dependent, because of a forbidden area near the cycloalkyl region.

1H-imidazo[4,5-c]quinolin-4-amines: novel non-xanthine adenosine antagonists.

On the basis of a model we recently developed for the antagonist binding site of the adenosine A1 receptor (J. Med. Chem. 1990, 33, 1708-1713), it was predicted that 1H-imidazo[4,5-c]quinolin-4-amines would be antagonists of the A1 receptor. Furthermore, it was expected that certain hydrophobic substitutions at the 2- and 4-positions would enhance affinity. Here, we report on the synthesis and the adenosine A1 and A2 receptor affinity of substituted 1H-imidazo[4,5-c]quinolin-4-amines. Some of these compounds have nanomolar affinity for the A1 receptor. The structure-activity relationships (SAR) of these compounds are discussed in relation to SAR for other adenosine receptor ligands. The 1H-imidazo[4,5-c]quinolin-4-amines constitute a novel class of non-xanthine adenosine antagonists.

A model for the antagonist binding site on the adenosine A1 receptor, based on steric, electrostatic, and hydrophobic properties.

With the aid of molecular modeling, both adenosine and adenosine A1 receptor antagonists belonging to various chemical classes were compared with respect to their minimum-energy conformations and molecular electrostatic potentials, as computed by the semiempirical molecular orbital program MOPAC. Distinct steric and electrostatic similarities between adenosine and the prototypic adenosine antagonist theophylline are evident when both compounds are superimposed, with theophylline in a so-called flipped orientation. Similar patterns were found for all other A1 antagonists investigated in this study. A model for the antagonist binding site on the adenosine A1 receptor, based on steric, electrostatic, and hydrophobic properties contributing to potency, is proposed.