Aliaa Abdelrahman

N-substituted phenoxazine and acridone derivatives: structure-activity relationships of potent P2X4 receptor antagonists.

P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was N-(benzyloxycarbonyl)phenoxazine (26, PSB-12054) with an IC(50) of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. N-(p-Methylphenylsulfonyl)phenoxazine (21, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC(50): 0.928-1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure-activity relationship analysis of P2X4 antagonists.

Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family

Adenine derivatives bearing substituents in the 2-, N(6)-, 7-, 8-, and/or 9-position and a series of deazapurines were synthesized and investigated in [(3)H]adenine binding studies at the adenine receptor in rat brain cortical membrane preparations (rAde1R). Steep structure-activity relationships were observed. Substitution in the 8-position (amino, dimethylamino, piperidinyl, piperazinyl) or in the 9-position (2-morpholinoethyl) with basic residues or introduction of polar substituents at the 6-amino function (hydroxy, amino, acetyl) represented the best modifications. Functional evaluation of selected adenine derivatives in adenylate cyclase assays at 1321N1 astrocytoma cells stably expressing the rAde1R showed that all compounds investigated were agonists or partial agonists. A subset of compounds was additionally investigated in binding studies at human embryonic kidney (HEK293) cells, which also express a high-affinity adenine binding site. Structure-affinity relationships at the human cell line were similar to those at the rAde1R, but not identical. In particular, N(6)-acetyladenine (25, K(i) rat: 2.85 microM; K(i) human: 0.515 microM) and 8-aminoadenine (33, K(i) rat: 6.51 microM; K(i) human: 0.0341 microM) were much more potent at the human as compared to the rat binding site. The new AdeR ligands may serve as lead structures and contribute to the elucidation of the functions of the adenine receptor family.

Extracellular NAD+ induces a rise in [Ca2+]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD(+)) is known to increase the intracellular calcium concentration [Ca(2+)](i) in different cell types and by various mechanisms. Here we show that NAD(+) triggers a transient rise in [Ca(2+)](i) in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca(2+) from IP(3)-responsive intracellular stores and an influx of extracellular Ca(2+). By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD(+)-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD(+). The identification of P2Y(1) and P2Y(11) as receptor subtypes responsible for the NAD(+)-triggered increase in [Ca(2+)](i) was supported by several lines of evidence. First, specific P2Y(1) and P2Y(11) receptor antagonists inhibited the NAD(+)-induced increase in [Ca(2+)](i). Second, NAD(+) was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD(+) caused an increase in [cAMP](i), prevented by the P2Y(11) receptor-specific antagonist NF157.

Cloning and Functional Expression of a Novel Gi Protein-Coupled Receptor for Adenine from Mouse Brain

An orphan G protein-coupled receptor from the rat has recently been demonstrated to act as a transmembrane receptor for the nucleobase adenine. The receptor is possibly involved in nociception. Here we report the cloning and functional expression of an additional Gi-coupled receptor for adenine (Genbank accession code DQ386867). mRNA for this receptor was obtained from mouse brain and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. The new mouse protein sequence shares only 76% identity with that of the rat adenine receptor, suggesting that the receptors are not species homologs but distinct receptor subtypes. In human 1321N1 astrocytoma cells stably expressing the new mouse receptor, adenine and 2-fluoroadenine inhibited the isoproterenol-induced cAMP formation with IC50 concentrations of 8 and 15 nM, respectively. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM) as well as the P2 receptor antagonist suramin (300 μM) failed to change the responses to adenine. In contrast, pretreatment of cells with pertussis toxin abolished the effect of adenine. When the novel adenine receptor was expressed in Sf21 insect cells, a specific binding site for [3H]adenine was detected. In competition assays, the rank order of potency of selected ligands was identical to that obtained in membranes from NG108-15 cells and rat brain cortex (adenine > 2-fluoroadenine > 7-methyladenine > 1-methyladenine ≫ N6-dimethyladenine). In summary, our data show that a second mammalian DNA sequence encodes for a Gi-coupled GPCR activated by low, nanomolar concentrations of adenine.

Interactions of Magnolia and Ziziphus extracts with selected central nervous system receptors

ETHNOPHARMACOLOGICAL RELEVANCE Magnolia officinalis Rehder and Wilson [Magnoliaceae] bark and Ziziphus spinosa (Buhge) Hu ex. Chen. [Fam. Rhamnaceae] seed have a history of use in traditional Asian medicine for mild anxiety, nervousness and sleep-related problems. AIM OF THE STUDY To identify pharmacological targets, extracts of Magnolia officinalis (ME), Ziziphus spinosa (ZE), and a proprietary fixed combination (MZE) were tested for affinity with central nervous system receptors associated with relaxation and sleep. METHODS In vitro radioligand binding and cellular functional assays were conducted on: adenosine A(1), dopamine (transporter, D(1), D(2S), D(3), D(4.4) and D(5)), serotonin (transporter, 5-HT(1A), 5-HT(1B), 5-HT(4e), 5-HT(6) and 5-HT(7)) and the GABA benzodiazepine receptor. RESULTS Interactions were demonstrated with the adenosine A(1) receptor, dopamine transporter and dopamine D(5) receptor (antagonist activity), serotonin receptors (5-HT(1B) and 5-HT(6) antagonist activity) and the GABA benzodiazepine receptor at a concentration of 100 microg/ml or lower. ME had an affinity with adenosine A(1) (K(i) of 9.2+/-1.1 microg/ml) and potentiated the GABA activated chloride current at the benzodiazepine subunits of the GABA receptor (maximum effect at 50 microg/ml). ME had a modest antagonist action with 5-HT(6) and ZE with the 5-HT(1B) receptor. CONCLUSION The interactions in the receptor binding models are consistent with the traditional anxiolytic and sleep-inducing activities of Magnolia officinalis bark and Ziziphus spinosa seed.

Development of Polar Adenosine A2A Receptor Agonists for Inflammatory Bowel Disease: Synergism with A2B Antagonists

Adenosine A2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects. 4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments. Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid and showed synergism with an A2B-selective antagonist. Thus, nonabsorbable, locally active A2A agonists, as a monotherapy or in combination with an A2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of known A2A agonists.

Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information

The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure–activity relationships within each ligand class, giving useful information for designing improved ligands.Graphical Abstract

Potent Suppressive Effects of 1-Piperidinylimidazole Based Novel P2X7 Receptor Antagonists on Cancer Cell Migration and Invasion.

The P2X7 receptor (P2X7R) has been reported as a key mediator in inflammatory processes and cancer invasion/metastasis. In this study, we report the discovery of novel P2X7R antagonists and their functional activities as potential antimetastatic agents. Modifications of the hydantoin core-skeleton and the side chain substituents of the P2X7R antagonist 7 were performed. The structure-activity relationships (SAR) and optimization demonstrated the importance of the sulfonyl group at the R1 position and the substituted position and overall size of R2 for P2X7R antagonism. The optimized novel analogues displayed potent P2X7 receptor antagonism (IC50 = 0.11-112 nM) along with significant suppressive effects on IL-1β release (IC50 = 0.32-210 nM). Moreover, representative antagonists (12g, 13k, and 17d) with imidazole and uracil core skeletons significantly inhibited the invasion of MDA-MB-231 triple negative breast cancer cells and cancer cell migration in a zebrafish xenograft model, suggesting the potential therapeutic application of these novel P2X7 antagonists to block metastatic cancer.

Carbamazepine derivatives with P2X4 receptor-blocking activity

Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives. The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC50 of 3.44μM). The present study extends the so far very limited knowledge on structure-activity relationships of P2X4 receptor antagonists.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe1103.24, Asn1153.29, Asn1734.60, Phe17945.39, Asn1945.40, Phe1955.41, Leu2015.47, His2526.54, and Tyr2687.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [3H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn1945.40 and Leu2015.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.