THE YEAST PLASMA-MEMBRANE PROTON-PUMPING ATPASE IS A VIABLE ANTIFUNGAL TARGET .1. EFFECTS OF THE CYSTEINE-MODIFYING REAGENT OMEPRAZOLE

The yeast plasma membrane proton pumping ATPase (H+-ATPase) was invest igated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent om eprazole on cell growth, glucose-induced medium acidification and HC-A TPase activity. Omeprazole inhibits the growth of Saccharomyces cerevi siae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of th e H+-ATPase and is fungicidal. Glucose-dependent medium acidification is correspondingly blocked by omeprazole and appears to require the H-ATPase to proceed through its reaction cycle, A strong correlation is observed between inhibition of medium acidification and H+-ATPase act ivity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated dru g with beta-mercaptoethanol, which is consistent with the expected for mation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C 867A) in the S. cerevisiae HC-ATPase suggests that covalent modificati on of the conserved C148 residue may be important for inhibition of AT Pase activity and cell growth. Other mutations (M128C and G158D/G156C) mapping near C148 support the importance of this region by modulating omeprazole inhibition of the H+-ATPase. These findings suggest that t he plasma membrane H+-ATPase may serve as an important molecular targe t for antifungal intervention.