Inhibition of gastric acid secretion is a major focus for therapy of acid-related diseases such as GERD, PUD and in combination with antibiotics, eradication of H. pylori. The gastric H,K-ATPase is a P2-type ATPase that catalyzes the electroneutral exchange of H+ for K+ in the secretory canaliculus membrane of the parietal cell. Since it catalyzes the final step of acid secretion it provides the best target for control of gastric acidity. PPIs are the mainstay of modern anti-acid therapy but these prodrugs have a short plasma half life and require the presence of significant acid secretion for activation and inhibition. Hence, they fall short in terms of speed of onset, control of symptoms and especially night time GERD in at least 20% of patients and also in eradication of Helicobacter pylori where nighttime acidity allows > 25% resistance to growth dependent antibiotics. The development of a safe H,K-ATPase inhibitor with rapid onset and long lasting inhibition without the requirement of acid activation requires detailed knowledge of the molecular mechanism of acid pumping. An Na,K-ATPase based homology model of the H,K-ATPase will be used to identify the K+ binding sites on the pump and the mechanism of ion occlusion and ion exchange. This will be facilitated by computer modeling of the docking site of a novel K+ competitive inhibitor. The inhibitor binding sites predicted by the model will be confirmed by site directed mutagenesis and be used to design and synthesize potent K+ competitive inhibitors. A key step in the regulation of acid secretion is the translocation of the ATPase from cytoplasmic tubulo-vesicles or tubule-cisternae to the microvilli of the secretory canaliculus and its association with a KCl conductance pathway. This must involve trafficking and sorting and interaction with a parietal cell protein scaffold. As an alternative to direct inhibition of the H,K-ATPase an understanding of the apical targeting of the pump and the induction of a KCl conductance required for acid secretion will enable a better description of the regulation of acid secretion and likely provide additional targets for long lasting control of acid secretion. H,K- ATPase sorting partners will be identified by immunoprecipitation and split-ubiquitin analysis and the results confirmed by siRNA silencing. A fluorescent proton pump inhibitor and a YFP-H,K-ATPase 2-subunit knock-in mouse model will be used to visualize in vivo and in real time, the transition of the pump from resting (tubulovesicles or tubulocisternae) to stimulated (canalicular) states of the secretory membrane of the parietal cell.
Adequate control of acid secretion, although much improved over the last two decades, is still elusive. In order to relieve continuing symptoms, damage and accelerate healing as well as provide a reliable and simple method of eradication of the gastric carcinogen, Helicobacter pylori. Several new approaches are planned such as long residence time Proton Pump Inhibitors (PPIs), long acting K+ competitive inhibitors and inhibition of sorting, trafficking and translocation pathways of the acid pump in the parietal cell.
Showing the most recent 10 out of 19 publications
