The yeast plasma membrane proton pumping ATPase is a viable antifungal target. I. Effects of the cysteine-modifying reagent omeprazole

The yeast plasma membrane proton pumping ATPase (H⁺-ATPase) was investigated as a potential molecular target for antifungal drug therapy by examining the inhibitory effects of the sulfhydryl-reactive reagent omeprazole on cell growth, glucose-induced medium acidification and H⁺-ATPase activity. Omeprazole inhibits the growth of Saccharomyces cerevisiae and the human pathogenic yeast Candida albicans in a pH dependent manner. Omeprazole action is closely correlated with inhibition of the 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 activity in plasma membranes isolated from treated cells. The inhibitory properties of omeprazole are blocked by pre-treatment of activated drug with β-mercaptoethanol, which is consistent with the expected formation of a sulfhydryl-reactive sulfenamide derivative. Mutagenesis of the three putative membrane sector cysteine residues (C148S, C312S, C867A) in the S. cerevisiae H⁺-ATPase suggests that covalent modification of the conserved C148 residue may be important for inhibition of ATPase 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 the plasma membrane H⁺-ATPase may serve as an important molecular target for antifungal intervention.