Studies on the mechanism of action of A80915A, a semi-naphthoquinone natural product, as an inhibitor of gastric (H(+)-K+)-ATPase.
A semi-naphthoquinone natural product, A80915A, produced by Streptomyces aculeolatus was found to be a potent inhibitor of gastric (H(+)-K+)-ATPase, the enzyme responsible for acid secretion in the stomach. Enzyme activity was measured by potassium-stimulated hydrolysis of ATP or p-nitrophenolphosphate with enzyme prepared from the stomach fundic mucosa of pigs. Concentration-dependent inhibition was observed with an IC50 of about 2-3 microM for both ATPase and p-nitrophenylphosphatase. A Hill plot indicated that the enzyme has two binding sites for A80915A. Inhibition was not affected by the presence of the reducing agent dithiothreitol, indicating a lack of involvement of enzyme sulfhydryl groups. A 30-min incubation of enzyme with increasing drug concentrations followed by a 10-fold dilution did not alter the IC50, indicating that A80915A does not covalently modify the enzyme. Coincubation of enzyme with 3.8 microM A80915A resulted in time-dependent inhibition. The rate of inhibition was slowed significantly by the presence of 20 mM potassium, rubidium and ammonium but not by 20 mM sodium, lithium and choline, or by 40 mM sucrose. The level of inhibition was influenced by the order of addition of potassium and drug to the enzyme. Taken together, these studies indicate that inhibition by A80915A is dependent on the conformation of gastric (H(+)-K+)-ATPase and that potassium slows the rate of inhibition by converting the enzyme to a conformation where the drug binding site is not as accessible. The mode of action of A80915A is distinct from that of two well characterized proton pump inhibitors, omeprazole and SCH 28080.
Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285.
A H
AH
Dantzig
P L
PL
Minor
J L
JL
Garrigus
D S
DS
Fukuda
J S
JS
Mynderse
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005753:Gastric Mucosa / Q000187:drug effects* / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D009285:Naphthoquinones / Q000494:pharmacology; D009853:Omeprazole / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D013552:Swine; D013997:Time Factors; D005723:gamma-Glutamyltransferase / Q000378:metabolism
C065210:A80915; D000900:Anti-Bacterial Agents; D007093:Imidazoles; D009285:Naphthoquinones; C035235:Sch 28080; C071895:A 80915A; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D009853:Omeprazole; D011188:Potassium; D004229:Dithiothreitol