A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation.

Enteric glial cells (EGC) modulate motility, maintain gut homeostasis, and contribute to neuroinflammation in intestinal diseases and motility disorders. Damage induces a reactive glial phenotype known as "gliosis", but the molecular identity of the inducing mechanism and triggers of "enteric gliosis" are poorly understood. We tested the hypothesis that surgical trauma during intestinal surgery triggers ATP release that drives enteric gliosis and inflammation leading to impaired motility in postoperative ileus (POI). ATP activation of a p38-dependent MAPK pathway triggers cytokine release and a gliosis phenotype in murine (and human) EGCs. Receptor antagonism and genetic depletion studies revealed P2X2 as the relevant ATP receptor and pharmacological screenings identified ambroxol as a novel P2X2 antagonist. Ambroxol prevented ATP-induced enteric gliosis, inflammation, and protected against dysmotility, while abrogating enteric gliosis in human intestine exposed to surgical trauma. We identified a novel pathogenic P2X2-dependent pathway of ATP-induced enteric gliosis, inflammation and dysmotility in humans and mice. Interventions that block enteric glial P2X2 receptors during trauma may represent a novel therapy in treating POI and immune-driven intestinal motility disorders.

Development of Anthraquinone Derivatives as Ectonucleoside Triphosphate Diphosphohydrolase (NTPDase) Inhibitors With Selectivity for NTPDase2 and NTPDase3.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of nucleoside tri- and di-phosphates to mono-phosphates. The products are subsequently hydrolyzed by ecto-5'-nucleotidase (ecto-5'-NT) to nucleosides. NTPDase inhibitors have potential as novel drugs, e.g., for the treatment of inflammation, neurodegenerative diseases, and cancer. In this context, a series of anthraquinone derivatives structurally related to the anthraquinone dye reactive blue-2 (RB-2) was synthesized and evaluated as inhibitors of human NTPDases utilizing a malachite green assay. We identified several potent and selective inhibitors of human NTPDase2 and -3. Among the most potent NTPDase2 inhibitors were 1-amino-4-(9-phenanthrylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (20, PSB-16131, IC50 of 539 nM) and 1-amino-4-(3-chloro-4-phenylsulfanyl)phenylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (48, PSB-2020, IC50 of 551 nM). The most potent NTPDase3 inhibitors were 1-amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (42, PSB-1011, IC50 of 390 nM) and 1-amino-4-(3-carboxy-4-hydroxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (33, PSB-2046, IC50 of 723 nM). The best NTPDase2 inhibitor 20 showed a non-competitive inhibition type, while the NTPDase3 inhibitor 42 behaved as a mixed-type inhibitor. These potent compounds were found to be selective vs. other NTPDases. They will be useful tools for studying the roles of NTPDase2 and -3 in physiology and under pathological conditions.

Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists.

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 µM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 µM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

Age-related shift in LTD is dependent on neuronal adenosine A2A receptors interplay with mGluR5 and NMDA receptors.

Synaptic dysfunction plays a central role in Alzheimer's disease (AD), since it drives the cognitive decline. An association between a polymorphism of the adenosine A2A receptor (A2AR) encoding gene-ADORA2A, and hippocampal volume in AD patients was recently described. In this study, we explore the synaptic function of A2AR in age-related conditions. We report, for the first time, a significant overexpression of A2AR in hippocampal neurons of aged humans, which is aggravated in AD patients. A similar profile of A2AR overexpression in rats was sufficient to drive age-like memory impairments in young animals and to uncover a hippocampal LTD-to-LTP shift. This was accompanied by increased NMDA receptor gating, dependent on mGluR5 and linked to enhanced Ca2+ influx. We confirmed the same plasticity shift in memory-impaired aged rats and APP/PS1 mice modeling AD, which was rescued upon A2AR blockade. This A2AR/mGluR5/NMDAR interaction might prove a suitable alternative for regulating aberrant mGluR5/NMDAR signaling in AD without disrupting their constitutive activity.

Ligand binding and activation of UTP-activated G protein-coupled P2Y2 and P2Y4 receptors elucidated by mutagenesis, pharmacological and computational studies.

The nucleotide receptors P2Y2 and P2Y4 are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to Gq proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y2R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, "drug-like" ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y2R appeared to be more spacious than that of P2Y4R. Mutation of Y197 to alanine in P2Y4R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y2- and P2Y4Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.

Antifibrotic and tumor microenvironment modulating effect of date palm fruit (Phoenix dactylifera L.) extracts in pancreatic cancer.

Date palm fruit (Phoenix dactylifera L.) is an endemic functional food, with great nutritional and economic importance due to its phytochemical compositions. The microenvironment of pancreatic cancer consists of cellular and acellular components, including fibroblasts, myofibroblasts, pancreatic stellate cells (PSCs), immune cells, blood vessels, extracellular matrix (ECM) and soluble proteins, such as cytokines and growth factors. The ECM represents a physical barrier that protects the tumor cell from active therapeutic compounds. In this study, four different solvents; water, ethanol, acetone, and ethyl acetate have been used to extract natural products from date palm fruit using a maceration method. The prepared extracts were investigated for antifibrotic (expression of fibronectin-1 and alpha-smooth muscle actin) and antiproliferative activity in tumor necrosis factor (TNF) stimulated PSCs in vitro. Based on the pharmacological test results, the ethyl acetate extract was subsequently partitioned into nine fractions based on polarity using silica gel column chromatography. These nine collective fractions were further evaluated for their activity. Ethanol, ethyl acetate and acetone, but not water extract significantly reduced PSC proliferation (p < 0.05). Date fruit fractions reduced fibrosis, decreased PSC activity and reversed the PSCs' fibrotic phenotype. The findings suggest a new approach for targeting pancreatic cancer through the modulation of PSC activity, thereby possibly enhancing the effect of known anticancer drugs. Moreover, date palm fruit appears to have chemopreventive activity protecting from pancreatic and probably other types of cancer, and thereby might be useful candidate to the pharmaceutical and nutraceutical industries in the development of natural compound-based industrial anticancer product.

Agonists and Antagonists for Purinergic Receptors.

Membrane receptors that are activated by the purine nucleoside adenosine (adenosine receptors) or by purine or pyrimidine nucleotides (P2Y and P2X receptors) transduce extracellular signals to the cytosol. They play important roles in physiology and disease. The G protein-coupled adenosine receptors comprise four subtypes: A1, A2A, A2B, and A3. The G-protein-coupled P2Y receptors are subdivided into eight subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, while the P2X receptors represent ATP-gated homomeric or heteromeric ion channels consisting of three subunits; the most important subunits are P2X1, P2X2, P2X3, P2X4, and P2X7. This chapter provides guidance for selecting suitable tool compounds for studying these large and important purine receptor families.

Memory deficits induced by chronic cannabinoid exposure are prevented by adenosine A2AR receptor antagonism.

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212-2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB1R) and cancelled by concomitant administration of adenosine A2A receptor (A2AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212-2 can also be reverted by A2AR antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212-2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212-2 (300 nM), an effect partially rescued by the A2AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A2AR or CB1R binding in the hippocampus and in the prefrontal cortex. These results, showing that A2AR antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A2ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.

A2B Adenosine Receptor Antagonists with Picomolar Potency.

The A2B adenosine receptor (A2BAR) was proposed as a novel target for the (immuno)therapy of cancer since A2BAR blockade results in antiproliferative, antiangiogenic, antimetastatic, and immunostimulatory effects. In this study, we explored the structure-activity relationships of xanthin-8-yl-benzenesulfonamides mainly by introducing a variety of linkers and substituents attached to the sulfonamide residue. A new, convergent strategy was established, which facilitated the synthesis of the target compounds. Many of the new compounds exhibited subnanomolar affinity for the A2BAR combined with high selectivity. Functional groups were introduced, which will allow the attachment of dyes and other reporter groups. 8-(4-((4-(4-Bromophenyl)piperazin-1-yl)sulfonyl)phenyl)-1-propylxanthine (34, PSB-1901) was the most potent A2B-antagonist ( Ki 0.0835 nM, KB 0.0598 nM, human A2BAR) with >10 000-fold selectivity versus all other AR subtypes. It was similarly potent and selective at the mouse A2BAR, making it a promising tool for preclinical studies. Computational studies predicted halogen bonding to contribute to the outstanding potency of 34.

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A2A receptor agonist (CGS21680), antagonist (KW6002) or the A1 receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A2A receptors modified the progressive loss of motor skills or survival of mSOD1G93A mice. Conversely, blockade of adenosine A1 receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

Antithrombotic P2Y12 receptor antagonists: recent developments in drug discovery.

The P2Y12 receptor is one of eight known P2Y receptor subtypes, and belongs to the G-protein-coupled receptor (GPCR) family. The P2Y12 receptor is highly expressed on blood platelets and in the brain. Potent, selective, peripherally acting antagonists for the P2Y12 receptor are used clinically as antithrombotic drugs. Several different scaffolds have been identified as P2Y12 receptor antagonists, including irreversibly acting thienotetrahydropyridines (prodrugs), and reversible competitive antagonists, including adenine nucleotide analogs, piperazinyl-glutamate-quinolines, -pyridines, and -pyrimidines, and anthraquinone derivatives. Here, we provide an overview of the different scaffolds that have been developed as P2Y12 receptor antagonists, some of which have become important therapeutics.

3-(2-Carboxyethyl)indole-2-carboxylic Acid Derivatives: Structural Requirements and Properties of Potent Agonists of the Orphan G Protein-Coupled Receptor GPR17.

The orphan receptor GPR17 may be a novel drug target for inflammatory diseases. 3-(2-Carboxyethyl)-4,6-dichloro-1 H-indole-2-carboxylic acid (MDL29,951, 1) was previously identified as a moderately potent GPR17 agonist. In the present study, we investigated the structure-activity relationships (SARs) of 1. Substitution of the indole 1-, 5-, or 7-position was detrimental. Only small substituents were tolerated in the 4-position while the 6-position accommodated large lipophilic residues. Among the most potent compounds were 3-(2-carboxyethyl)-1 H-indole-2-carboxylic acid derivatives containing the following substituents: 6-phenoxy (26, PSB-1737, EC50 270 nM), 4-fluoro-6-bromo (33, PSB-18422, EC50 27.9 nM), 4-fluoro-6-iodo (35, PSB-18484, EC50 32.1 nM), and 4-chloro-6-hexyloxy (43, PSB-1767, EC50 67.0 nM). (3-(2-Carboxyethyl)-6-hexyloxy-1 H-indole-2-carboxylic acid (39, PSB-17183, EC50 115 nM) behaved as a partial agonist. Selected potent compounds tested at human P2Y receptor subtypes showed high selectivity for GPR17. Docking into a homology model of the human GPR17 and molecular dynamic simulation studies rationalized the observed SARs.

Beneficial Effect of a Selective Adenosine A2A Receptor Antagonist in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease.

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) and mitigates both amyloid and Tau lesions in transgenic mouse models of the disease. While short-term treatment with A2AR antagonists have been shown to alleviate cognitive deficits in mouse models of amyloidogenesis, impact of a chronic and long-term treatment on the development of amyloid burden, associated neuroinflammation and memory deficits has never been assessed. In the present study, we have evaluated the effect of a 6-month treatment of APPsw/PS1dE9 mice with the potent and selective A2AR antagonist MSX-3 from 3 to 9-10 months of age. At completion of the treatment, we found that the MSX-3 treatment prevented the development of memory deficits in APP/PS1dE9 mice, without significantly altering hippocampal and cortical gene expressions. Interestingly, MSX-3 treatment led to a significant decrease of Aß1-42 levels in the cortex of APP/PS1dE9 animals, while Aß1-40 increased, thereby strongly affecting the Aß1-42/Aß1-40 ratio. Together, these data support the idea that A2AR blockade is of therapeutic value for AD.

Treatment with A2A receptor antagonist KW6002 and caffeine intake regulate microglia reactivity and protect retina against transient ischemic damage.

Transient retinal ischemia is a major complication of retinal degenerative diseases and contributes to visual impairment and blindness. Evidences indicate that microglia-mediated neuroinflammation has a key role in the neurodegenerative process, prompting the hypothesis that the control of microglia reactivity may afford neuroprotection to the retina against the damage induced by ischemia-reperfusion (I-R). The available therapeutic strategies for retinal degenerative diseases have limited potential, but the blockade of adenosine A2A receptor (A2AR) emerges as candidate strategy. Therefore, we evaluated the therapeutic potential of a selective A2AR antagonist (KW6002) against the damage elicited by I-R. The administration of KW6002 after I-R injury reduced microglia reactivity and inflammatory response and afforded protection to the retina. Moreover, we tested the ability of caffeine, an adenosine receptor antagonist, in mediating protection to the retina in the I-R injury model. We demonstrated that caffeine administration dually regulated microglia reactivity and cell death in the transient retinal ischemic model, depending on the reperfusion time. At 24 h of reperfusion, caffeine increased microglial reactivity, inflammatory response and cell death elicited by I-R. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced the levels of proinflammatory cytokines and cell death. Together, these results provide a novel evidence for the use of adenosine A2AR antagonists as potential therapy for retinal ischemic diseases and demonstrate the effect of caffeine on the regulation of microglia-mediated neuroinflammation in the transient ischemic model.

Molecular Recognition of Agonists and Antagonists by the Nucleotide-Activated G Protein-Coupled P2Y2 Receptor.

A homology model of the nucleotide-activated P2Y2R was created based on the X-ray structures of the P2Y1 receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design.

Date Palm Tree (Phoenix dactylifera L.): Natural Products and Therapeutic Options.

Many plants, including some of the commonly consumed herbs and spices in our daily food, can be safely and effectively used to prevent and/or treat some health concerns. For example, caffeine the active ingredient found in coffee beans (Coffea), shows biological activity in the treatment of the central nervous system (CNS) disorders, indole-3-carbinol, and 3,3'-diindolylmethane are both broccoli (Brassica oleracea) derived phytochemicals with potential anti-cancer activity, and resveratrol, isolated from grape (Vitis vinifera), is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects. Date palm fruits possess high nutritional and therapeutic value with significant antioxidant, antibacterial, antifungal, and anti-proliferative properties. This review focuses on the date fruit extracts and their benefits in individual health promoting conditions and highlights their applications as useful to the pharmaceutical and nutraceutical industries in the development of natural compound-based industrial products.

Development of Potent and Selective Antagonists for the UTP-Activated P2Y4 Receptor.

P2Y4 is a Gq protein-coupled receptor activated by uridine-5'-triphosphate (UTP), which is widely expressed in the body, e.g., in intestine, heart, and brain. No selective P2Y4 receptor antagonist has been described so far. Therefore, we developed and optimized P2Y4 receptor antagonists based on an anthraquinone scaffold. Potency was assessed by a fluorescence-based assay measuring inhibition of UTP-induced intracellular calcium release in 1321N1 astrocytoma cells stably transfected with the human P2Y4 receptor. The most potent compound of the present series, sodium 1-amino-4-[4-(2,4-dimethylphenylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-16133, 61) exhibited an IC50 value of 233 nM, selectivity versus other P2Y receptor subtypes, and is thought to act as an allosteric antagonist. A receptor homology model was built and docking studies were performed to analyze ligand-receptor interactions. Compound 64 (PSB-1699, sodium 1-amino-4-[4-(3-pyridin-3-ylmethylthio)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate) represents the most selective P2Y4 receptor antagonist known to date. Compounds 61 and 64 are therefore anticipated to become useful tools for studying this scarcely investigated receptor.

Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation.

Cannabinoid-mediated memory impairment is a concern in cannabinoid-based therapies. Caffeine exacerbates cannabinoid CB1 receptor (CB1R)-induced memory deficits through an adenosine A1 receptor-mediated mechanism. We now evaluated how chronic or acute blockade of adenosine A2A receptors (A2ARs) affects long-term episodic memory deficits induced by a single injection of a selective CB1R agonist. Long-term episodic memory was assessed by the novel object recognition (NOR) test. Mice received an intraperitoneal (i.p.) injection of the CB1/CB2 receptor agonist WIN 55,212-2 (1 mg/kg) immediately after the NOR training, being tested for novelty recognition 24 h later. Anxiety levels were assessed by the Elevated Plus Maze test, immediately after the NOR. Mice were also tested for exploratory behaviour at the Open Field. For chronic A2AR blockade, KW-6002 (istradefylline) (3 mg/kg/day) was administered orally for 30 days; acute blockade of A2ARs was assessed by i.p. injection of SCH 58261 (1 mg/kg) administered either together with WIN 55,212-2 or only 30 min before the NOR test phase. The involvement of CB1Rs was assessed by using the CB1R antagonist, AM251 (3 mg/kg, i.p.). WIN 55,212-2 caused a disruption in NOR, an action absent in mice also receiving AM251, KW-6002 or SCH 58261 during the encoding/consolidation phase; SCH 58251 was ineffective if present during retrieval only. No effects were detected in the Elevated Plus maze or Open Field Test. The finding that CB1R-mediated memory disruption is prevented by antagonism of adenosine A2ARs, highlights a possibility to prevent cognitive side effects when therapeutic application of CB1R drugs is desired.

Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors.

Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A1/A2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A1 and A2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0-1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0-60.0 mg/kg), and even blocked social preference at higher doses (30.0-60.0 mg/kg). The A1 antagonist Cyclopentyltheophylline (CPT; 3-9 mg/kg) did not modify social contact or preference on its own, and the A2A antagonist MSX-3 (1.5-6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5-1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0-30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol impairs the formation of social memories, and A2A adenosine antagonists can prevent the amnestic effects of ethanol, so that animals can recognize familiar conspecifics. On the other hand, ethanol can counteract the social withdrawal induced by caffeine, a non-selective adenosine A1/A2A receptor antagonist. These results show the complex set of interactions between ethanol and caffeine, some of which could be the result of the opposing effects they have in modulating the adenosine system.

Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila.

Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires' disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC50 values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC50-value of 4.24µM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.