Generation and deposition of the amyloid b (Ab) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and c-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Ab, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits b-cleavage of APP and reduces levels of secreted and intracellular Ab in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Ab in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases b-cleavage of APP and results in a significant reduction in the level of Ab40 and Ab42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Ab. This pivotal first report of central Ab lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.

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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