Characterization of a novel potassium-competitive acid blocker of the gastric H,K-ATPase, 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate (TAK-438)

Abstract

Inhibition of the gastric H,K-ATPase by the potassium-competitive acid blocker (P-CAB) 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine (TAK-438), is strictly K(+)-competitive with a K(i) of 10 nM at pH 7. In contrast to previous P-CABs, this structure has a point positive charge (pK(a) 9.06) allowing for greater accumulation in parietal cells compared with previous P-CABs [e.g., (8-benzyloxy-2-methyl-imidazo(1,2-a)pyridin-3-yl)acetonitrile (SCH28080), pK(a) 5.6]. The dissociation rate of the compound from the isolated ATPase is slower than other P-CABs, with the t(1/2) being 7.5 h in 20 mM KCl at pH 7. The stoichiometry of binding of TAK-438 to the H,K-ATPase is 2.2 nmol/mg in the presence of Mg-ATP, vanadate, or MgP(i). However, TAK-438 also binds enzyme at 1.3 nmol/mg in the absence of Mg(2+). Modeling of the H,K-ATPase to the homologous Na,K-ATPase predicts a close approach and hydrogen bonding between the positively charged N-methylamino group and the negatively charged Glu795 in the K(+)-binding site in contrast to the planar diffuse positive charge of previous P-CABs. This probably accounts for the slow dissociation and high affinity. The model also predicts hydrogen bonding between the hydroxyl of Tyr799 and the oxygens of the sulfonyl group of TAK-438. A Tyr799Phe mutation resulted in a 3-fold increase of the dissociation rate, showing that this hydrogen bonding also contributes to the slow dissociation rate. Hence, this K(+)-competitive inhibitor of the gastric H,K-ATPase should provide longer-lasting inhibition of gastric acid secretion compared with previous drugs of this class.

Fig. 1.

Structure of TAK-438.

Fig. 2.

Inhibition by TAK-438. A, an aliquot of the gastric H,K-ATPase enzyme suspension (3 μg/ml) was preincubated in a buffer composed of 10 mM KCl, 2 mM MgCl₂, ± 2 μg/ml nigericin, and 20 mM Tris/HCl, pH 7.4, for 2 h, and the enzyme activity was measured by adding 2 mM ATP for 30 min at 37°C. B, 1/V versus 1/[KCl] plot in the presence of different fixed concentrations of TAK-438. V represents the enzyme activity (μmol P_i · mg⁻¹ · h⁻¹). [KCl] represents a concentration of KCl (mM). Each point represents mean ± S.E. of three experiments.

Fig. 3.

The enzyme suspension (20 μg/ml) in a buffer composed of 3 mM PIPES/Tris, pH 7, 2 mM MgCl₂, 150 mM KCl, 5 μg/ml valinomycin, and 1 μM acridine orange (AO) was preincubated at 37°C in a cell of a spectrofluorimeter for 5 min. After 60 s, ATP (2 mM) was added to initiate acridine orange uptake. The inhibitor was added at the time as indicated by P-CAB. SCH and TAK represent SCH28080 and TAK-438, respectively.

Fig. 4.

Dissociation of TAK-438 binding by KCl at different incubation times. A, enzyme (10 μg/ml) was resuspended in a buffer composed of 50 mM Tris/HCl, pH 7.0, 2 mM MgCl₂, 2 mM ATP, and 50 nM [¹⁴C]TAK-438 and incubated at room temperature (25°C) for 60 min. Under this condition, TAK-438 binds to the enzyme with full inhibition. To this enzyme suspension, KCl was added at 10 or 20 mM and incubated at 37°C. At timed intervals, an aliquot was taken to measure the binding. B, enzyme (10 μg/ml) was resuspended in a buffer composed of 50 mM Tris/HCl, pH 7.0, 2 mM MgCl₂, 2 mM ATP, and 100 nM [¹⁴C]TAK-438 and incubated at room temperature (25°C) for 60 min. Under these conditions, TAK-438 binds to the enzyme with full inhibition. To this enzyme suspension, KCl was added up to 0.3 M final concentration and incubated at 37°C. At timed intervals, an aliquot was taken to measure the binding as described under Materials and Methods.

Fig. 5.

Dissociation of TAK-438 binding as a function of KCl concentration and medium pH. A, enzyme (5 μg/ml) was resuspended in a buffer composed of 2 mM MgCl₂, 2 mM ATP, 2 μg of nigericin/ml, 50 nM [¹⁴C]TAK-438, and 50 mM Tris/HCl, pH 7.0. The enzyme suspension at pH 7 was incubated at 37°C for 3 h in the presence of various concentrations of KCl. B, the enzyme suspensions at pH 6.1 and 8 were treated identically except for incubation time and temperature. These enzyme suspensions were incubated at 25°C for 2 h. TAK-binding was measured as described under Materials and Methods. In this experiment, TAK-438 binding measured in the absence of KCl was 2.15 nmol/mg at 50 nM TAK-438. This was taken as representing 100% binding, allowing calculation of the percentage of TAK-438 binding at given KCl concentrations.

Fig. 6.

Exchange of bound [¹⁴C]TAK-438 by unbound unlabeled TAK-438 after different incubation times. Enzyme (10 μg/ml) was resuspended in a buffer composed of 50 mM Tris/HCl, pH 7.0, 2 mM MgCl₂, 2 mM ATP, and 100 nM [¹⁴C]TAK-438 and incubated at room temperature (25°C) for 60 min. Under these conditions, TAK-438 binds to the enzyme with full inhibition. To this enzyme suspension, nonlabeled TAK-438 was added up to 50 μM final concentration and incubated at 37°C. At timed intervals, an aliquot was taken to measure the exchange as described under Materials and Methods.

Fig. 7.

Effects of various ligands on TAK-438 binding. The gastric vesicles (0.01 mg/ml) were incubated at 37°C for 60 min in a buffer composed of 50 mM Tris/HCl, pH 7.0, and different ligands such as ± 2 mM MgCl₂, ± 5 mM CDTA, ± 2 mM ATP, ± 0.2 mM vanadate, and ± 5 mM P_i/Tris, pH 7.0, in the presence of [¹⁴C]TAK-438 (100 nM). Binding stoichiometry was determined as described under Materials and Methods.

Fig. 8.

Ribbon representation of the H,K-ATPase model based on the crystallographic structure of the Na,K-ATPase, PDB 2ZXE. The major domains are noted, and the position of the membrane is indicated with yellow lines: TM1, dark blue; TM2, cyan; TM3, light green; TM4, green-blue; TM5, light yellow; TM6, dark yellow; TM7, light brown; TM8, dark brown; TM9, light red; TM10, dark red. This color scheme is maintained in all figures. The position of the ion binding site is close to the middle of the membrane (ball and stick). The inhibitor, TAK-438 (space filling in light blue), binds ∼10 Å from the ion binding sight in a cavity bounded by TM1, TM2, TM4, and the TM5-TM6 loop. The N-terminal 27 residues of the β subunit (pink ribbon) are not present in the Na,K-ATPase structure PDB 2ZXE and therefore are not in the HKzxe model.

Fig. 9.

A, SCH28080 binding to the H,K-ATPase model based on PDB 2ZXE. Binding is in the space between Ala335 and Cys813 (green and yellow spheres, respectively). TM1 and TM2 helices (light blue and blue, respectively) enclose the site to the right providing interaction between the inhibitor and Cys120, Asn138, Leu141, and Asp137 (stick). There is also a closer approach by the inhibitor to Pro798 (TM8) and Met334 (TM4) than in previously published models. The binding site is stabilized from below by aromatic interactions between Tyr799 and Phe332 (below Ala335 in TM4; not shown for clarity). B, binding of TAK-438 to the H,K-ATPase. The inhibitor is predicted to bind in the same cleft as SCH28080. Hydrogen bonding between Tyr799 and the sulfonyl oxygens of the inhibitor and the proximity of its amino group to the ion binding site are predicted to contribute to the slow off rate for TAK-438.