title: A model of reversible inhibitors in the gastric H+/K+-ATPase binding site determined by rotational echo double resonance NMR.
abstract: 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 IC(50) values for porcine H(+)/K(+)-ATPase inhibition falling in the sub-10 microm range. Deuterium NMR spectra of a (2)H-labeled inhibitor titrated into H(+)/K(+)-ATPase membranes revealed that 80-100% of inhibitor was bound to the protein, and K(+)-competition (2)H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from (13)C-(19)F 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 Phe(126) 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.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: J A
authors.initials: JA
authors.lastname: Watts
authors.forename: A
authors.initials: A
authors.lastname: Watts
authors.forename: D A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005461:Fluorine / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054328:Proton Pump Inhibitors; D011522:Protons; D013270:Stomach / Q000201:enzymology*; D013552:Swine
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D054328:Proton Pump Inhibitors; D011522:Protons; C035235:Sch 28080; D005461:Fluorine; D010649:Phenylalanine; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase