H+/K+ competition in the inhibition of the H,K ATPase by acid pump antagonists

GIULIANA A. FERNANDEZ; SANTIAGO E. FARAJ; MÓNICA R. MONTES; NICOLE T. CERF; ROLANDO C. ROSSI

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

The gastric H,K-ATPase is a membrane protein located in the parietal cells of the stomach, responsible for acidifying gastric juice. Driven by ATP hydrolysis, the H,K ATPase couples H+ extrusion to the uptake of K+. The reaction cycle of the H,K ATPase includes the interconversion between two main states of the enzyme, E1 and E2. The ion binding sites alternate between being exposed toward the cytoplasmic side (E1, with high affinity for H+) and the extracellular side (E2, with high affinity for K+). The H,K-ATPase is the molecular target of different drugs for the treatment of diseases related to acid secretion. The presence of a hydrophobic pocket exposed to the extracellular medium was determined from the analysis of the protein structure. Such a pocket has been proposed as the potential interaction site of compounds belonging to two families of inhibitors: the classic proton pump inhibitors (PPIs) and the acid pump antagonists (APAs), recently marketed in Asia. This work aims to evaluate the effect of [H+] on the competition of K+ and APAs (vonoprazan and tegoprazan) for the binding to the extracellular side of the gastric pump. We measured enzyme activity and the kinetics of E1–E2 conformational changes using a non-compartmentalized preparation in order to evaluate the conditions that increase the potency of APAs. To explore the contribution of individual amino acids to ligand binding, we performed a prediction of pKa values in both states.We found that APAs bind preferentially to the luminal-facing E2 state; binding to the cytoplasmic side of the enzyme also occurs, even though with low affinity. Data were fitted by non-linear regression to a minimal model that adequately describes the interaction between the H,K-ATPase, APAs and K+ at different pH. Our results suggest that the competition between K+ and H+ could explain the increased potency of APAs at acidic pH, as an alternative to the protonation of the inhibitors, which is currently the only proposed explanation.