title: Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase.
abstract: 1. FPL 67156 (6-N,N-diethyl-beta, gamma-dibromomethylene-D-ATP), is a newly synthesized analogue of ATP. 2. In a rabbit isolated tracheal epithelium preparation, measuring P2U-purinoceptor-dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP-gamma-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5-fold in the presence of 100 microM FPL 67156. The differential effect of FPL 67156 on UTP and ATP-gamma-S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3. FPL 67156 was tested as an ecto-ATPase inhibitor in a human blood cell assay, measuring [gamma 32P]-ATP dephosphorylation. The compound inhibited [gamma 32P]-ATP degradation with a pIC50 of 4.6. 4. FPL 67156 was then tested for its effects on ATP and alpha, beta-methylene-ATP responses at P2X-purinoceptors in the rabbit isolated ear artery. In the concentration range 30 microM-1 mM, the compound potentiated the contractile effects of ATP but not those of alpha, beta-methylene-ATP. At 1 mM, FPL 67156 produced a 34-fold leftward shift of ATP E/[A] curves. 5. The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analyzed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pKi for FPL 67156 as an ecto-ATPase inhibitor of 5.2. 6. Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X- and P2T-purinoceptors (pA2 ~ 3.3 and 3.5 respectively), and weak agonist effects at P2u-purinoceptors(p[A 50]~ 3.5).7. The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto-ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors.8.This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.
authors.affiliation: Department of Pharmacology, Fisons R&D Labs, Loughborough, Leicestershire.
authors.forename: B E
authors.initials: BE
authors.lastname: Crack
authors.forename: C E
authors.initials: CE
authors.lastname: Pollard
authors.forename: M W
authors.initials: MW
authors.lastname: Beukers
authors.forename: S M
authors.initials: SM
authors.lastname: Roberts
authors.forename: S F
authors.initials: SF
authors.lastname: Hunt
authors.forename: A H
authors.initials: AH
authors.lastname: Ingall
authors.forename: K C
authors.initials: KC
authors.lastname: McKechnie
authors.forename: A P
authors.initials: AP
authors.lastname: IJzerman
authors.forename: P
authors.initials: P
authors.lastname: Leff
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002712:Chlorides / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008297:Male; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011817:Rabbits; D018048:Receptors, Purinergic P2 / Q000187:drug effects; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D002712:Chlorides; D007473:Ion Channels; D018048:Receptors, Purinergic P2; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D014544:Uridine Triphosphate