A Model of Reversible Inhibitors in the Gastric H+/K +-ATPase Binding Site Determined by Rotational Echo Double Resonance NMR

Several close analogues of the noncovalent H+/K +-ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC50 values for porcine H+/K+-ATPase inhibition falling in the sub-10 μM range. Deuterium NMR spectra of a 2H-labeled inhibitor titrated into H+/K +-ATPase membranes revealed that 80-100% of inhibitor was bound to the protein, and K+-competition 2H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from 13C-19F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H+/K +-ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe126 close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.