Background and purpose: Antagonist effects at the P2X 7 receptor are complex with many behaving in a non-competitive manner. In this study, the effects of N- [2-({2-[(2-hydroxyethyl) ylacetamide (compound-17) and N 2 -(3,4-difluorophenyl)-N 1 -[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride (GW791343) on P2X 7 receptors were examined and their mechanism of action explored. Experimental approach: Antagonist effects were studied by measuring agonist-stimulated ethidium accumulation in cells expressing human or rat recombinant P2X 7 receptors and in radioligand binding studies. Key results: Compound-17 and GW791343 were non-competitive inhibitors of human P2X 7 receptors. Receptor protection studies using decavanadate and pyridoxalphosphate-6-azophenyl-2',4 0 -disulphonic acid (PPADS) showed that neither compound-17 nor GW791343 competitively interacted at the ATP binding site and so were probably negative allosteric modulators of the P2X 7 receptor. GW791343 prevented the slowly reversible blockade of the human P2X 7 receptor produced by compound-17 and inhibited [ 3 H]-compound-17 binding to the P2X 7 receptor suggesting they may bind to similar or interacting sites. At rat P2X 7 receptors, compound-17 was a negative allosteric modulator but the predominant effect of GW791343 was to increase agonist responses. Antagonist interaction and radioligand binding studies revealed that GW791343 did not interact at the ATP binding site but did interact with the compound-17 binding site suggesting that GW791343 is a positive allosteric modulator of the rat P2X 7 receptor. Conclusions: Compound-17 was a negative allosteric modulator of human and rat P2X 7 receptors. GW791343 was a negative allosteric modulator of the human P2X 7 receptor but at the rat P2X 7 receptor its predominant effect was positive allosteric modulation. These compounds should provide valuable tools for mechanistic studies on P2X 7 receptors.

Background and Purpose: The P2X 7 receptor exhibits complex pharmacological properties. In this study, binding of a [ 3 H]-labelled P2X 7 receptor antagonist to human P2X 7 receptors has been examined to further understand ligand interactions with this receptor. -ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X 7 receptors. Key Results: Binding of [ 3 H]-compound-17 was higher in membranes prepared from cells expressing P2X 7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X 7 receptors. Binding was reversible, saturable and modulated by P2X 7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation.Conclusions: These data demonstrate that human P2X 7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X 7 receptor complex enhances subsequent binding to other P2X 7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X 7 receptor.

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Background and purpose: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1b release in dog and human whole blood. Key results: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.

Reaction of vinyl-fl-ionol (1) [6] with the lithium or sodium salt of a sulfinic acid, essentially as described by Julia [5], gave the Cl5-sulfones 2 (a-k) 1) which on alkylation with l-acetoxy-3-chlormethyl-2-butene (3) [7] afforded the Czo-sulfones 4 (a-i) ; certain of these on base treatment furnished vitamin A (5). Table I lists the C15-and Czo-sulfones which were used in the present study.It was recognized at the onset that a major problem in this synthesis would be the elimination of the sulfinic acid from the Czo-sulfone. To this end we have explored some of the factors which were expected to influence the elimination reaction, namely electronic and steric factors associated with the sulfone group, as well as the effect of various bases and solvents.Preparation of Cis-Sulfones. -The C15-sulfones 2 were conveniently prepared[5] by reacting vinyl-fl-ionol (1) with the sodium or lithium salt of the sulfinic acid [S] in acetic acid. In general there was a rapid disappearance of starting material with the initial formation of several products, however, as the reaction proceeded, one of these was further converted into the all-tram primary sulfone which becomes the main product. Scheme 2 8 I 9By terminating the reaction at an early stage it was possible to isolate the primary all-trans-and Ag.10-cis-sulfones, 2 and 6 respectively (see Scheme 2). In addition, the 1)Numbering system: CIS and CZO refer to the isoprenoid skeleton.