Ro 18-5364, a potent new inhibitor of the gastric (H+ + K+)-ATPase

The sulfoxide agent Ro 18-5364 is an extremely potent and rapid inhibitor of the gastric mucosal (H+ + K+)-ATPase with an apparent Ki of 0.1 microM at pH 6. The inhibition of both enzymatic activity and vesicular proton transport in membrane preparations is concentration- and time-dependent. Comparative studies with the two enantiomers of Ro 18-5364 indicated no enantiomeric preference. Marked differences were seen between Ro 18-5364 (sulfoxide) and Ro 18-5362 (sulfide) with regard to inhibitory activity. Even at concentrations as high as 0.1 mM Ro 18-5362 failed to affect significantly (H+ + K+)-ATPase activity and associated proton translocation. Similarly, Ro 17-5380 demonstrated an apparent Ki of 20 microM for inhibition of the (H+ + K+)-ATPase whereas its reduced derivative Ro 17-4749 was inactive. Addition of a single methyl group in the pyridine moiety of Ro 18-5364 noticeably decreased the inhibitory potency. The inhibitory action of Ro 18-5364 on (H+ + K+)-ATPase activity was markedly higher at low incubation medium pH in comparison to physiological or alkaline values. The results of incorporation studies paralleled that of enzymatic inhibition. The extent of Ro 18-5364 incorporation was dependent on time, concentration, and medium hydrogen ion concentration, with a decrease in medium pH resulting in increased binding. While ATP and GTP had little effect on the binding rates, reduced lipoic acid methyl ester, mercaptoethanol and dithiothreitol were capable of displacing the radiolabel to different extents. Autoradiography of electrophoresed Ro-18-5364-labeled gastric microsomal membranes confirmed that the radiolabel was associated with polypeptides of approximately 100 kDa. The incorporation was reversed upon subjection of the membranes to reducing conditions.