Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto‐ATPase

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Abstract
1 FPL 67156 (6-N,N-diethyl-β,γ-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-γ-S. UTP agonist-concentration effect (E/[A]) curves were shifted to the left by 5-fold in the presence of 100 μm FPL 67156. The differential effect of FPL 67156 on UTP and ATP-γ-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 [γ32P]-ATP dephosphorylation. The compound inhibited [γ32P]-ATP degradation with a pIC50 of 4.6. 4 FPL 67156 was then tested for its effects on ATP and α,β-methylene-ATP responses at P2X-purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μm-1 mm, the compound potentiated the contractile effects of ATP but not those of α,β-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 analysed 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 pK1 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[A50] ≅ 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.

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