Hyperexpression of the

Cationic peptides produced by multicellular organisms are an evolutionarily ancient and rapidly mobilized primary defense against infections caused by a broad range of microbes (8). The cationic antimicrobial peptides are ribosomally synthesized, proteolytically processed species of 12 to ϳ50 amino acids that comprise about 50% hydrophobic residues and that have a net excess of positive charge (9). They are usually found on epithelial cell surfaces and in phagocytic cells at sites of microbial invasion, and only a few instances of constitutive or induced resistance to cationic peptides have been detected (for a review, see reference 44). Although the cationic peptides are subdivided into several structural classes (10), they are, in general, amphipathic molecules that preferentially bind to acidic phospholipids, acidic polysaccharides, and lipopolysaccharides on the exterior of the lipid bilayer of invading microbes rather than to the cholesterol-rich and neutral plasma membrane surfaces of mammalian host cells. The bound cationic peptides are then thought to kill target microbes, including fungi (4), by forming assemblies that alter the lipid bilayer structure and disrupt the functional properties of the microbial membrane. A few cationic peptides may affect intracellular targets, including mitochondria and DNA and RNA metabolism; but apart from the binding of the salivary histatin 5 to a cell surface receptor in Candida albicans (16), there is no evidence of direct effects on fungal cell surface proteins. We hypothesized that the incorporation of a cationic peptide-like motif into an antifungal would enhance its potency by concentrating the compound at fungal cell surfaces. This idea has been validated in the present study by obtaining a membraneimpermeant and surface-active cationic peptide inhibitor of the fungal plasma membrane proton-pumping ATPase (Pma1p), an essential enzyme involved in fungal energy transduction (36).Pma1p is an ϳ100-kDa electrogenic, polytopic integral membrane protein of the P-type ATPase class which contributes 10 to 20% of the yeast plasma membrane protein. It generates the plasma membrane electrochemical gradient that is required for the maintenance of intracellular pH, cellular ion balance, and the uptake of numerous nutrients (36). The amount of functional Pma1p is tightly regulated (5), and yeast growth requires at least 25% of normal Pma1p activity (35). Pma1p was postulated to be a target for surface-mediated, broad-spectrum antifungal intervention because of the structural similarity between cell surface loops in Pma1ps from fungal cells and their dissimilarity to the comparable loops in P-type ATPases from other organisms, as well as the specificity achieved with therapies targeting mammalian P-type ATPases (28). Pma1p was validated as an antifungal target by demonstrating that acid-activated omeprazole is a fungicidal Pma1p inhibitor that acts from outside the cell (25,37). This paper describes a drug discovery strategy that targets Pma1p. Screen-* Corresponding author. Mai...