title: Mechanism of action of species-selective P2X(7) receptor antagonists.
abstract: OBJECTIVE
  AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X(7) receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues.
  
  
  METHODS
  Antagonist effects at recombinant and chimeric P2X(7) receptors were assessed by ethidium accumulation and radioligand-binding studies.
  
  
  RESULTS
  AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X(7) receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X(7) receptors and, for each compound, their potency estimates at human and dog P2X(7) receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X(7) receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X(7) receptor. N(2)-(3,4-difluorophenyl)-N(1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373.
  
  
  CONCLUSIONS
  AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X(7) receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X(7) receptor at which antagonists can bind and affect receptor function.
authors.affiliation: Neurology Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Essex, UK. anton.d.michel@hotmail.co.uk
authors.forename: Anton D
authors.initials: AD
authors.lastname: Michel
authors.forename: Sin-Wei
authors.initials: SW
authors.lastname: Ng
authors.forename: Shilina
authors.initials: S
authors.lastname: Roman
authors.forename: William C
authors.initials: WC
authors.lastname: Clay
authors.forename: David K
authors.initials: DK
authors.lastname: Dean
authors.forename: Daryl S
authors.initials: DS
authors.lastname: Walter
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D053961:Azabicyclo Compounds / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D010879:Piperazines / Q000494:pharmacology; D011487:Protein Conformation; D058919:Purinergic P2 Receptor Antagonists; D011869:Radioligand Assay; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology*; D014161:Transduction, Genetic; D014638:Vanadates / Q000494:pharmacology
chemical_list: C516674:AZ 11645373; D053961:Azabicyclo Compounds; C528279:GW791343; C546022:N-(2-methyl-5-(9-oxa-3,7-diazabicyclo(3.3.1)non-3-ylcarbonyl)phenyl)-2-tricyclo(3.3.1.13,7)dec-1-ylacetamide; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D010879:Piperazines; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013844:Thiazoles; D014638:Vanadates; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000218:Adamantane; D005998:Glycine