Surface-Active Fungicidal d -Peptide Inhibitors of the Plasma Membrane Proton Pump That Block Azole Resistance

Abstract

ABSTRACT A 1.8-million-member d -octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif. The C-terminal motif concentrated the library members at the fungal cell surface. A primary screen for inhibitors of Saccharomyces cerevisiae and Candida albicans growth, together with an in vitro secondary screen with the S. cerevisiae plasma membrane ATPase (Pma1p) as a target, identified the antifungal d -octapeptide BM0 ( d -NH 2 -RFWWFRRR-CONH 2 ). Optimization of BM0 led to the construction of BM2 ( d -NH 2 -RRRFWWFRRR-CONH 2 ), which had broad-spectrum fungicidal activity against S. cerevisiae , Candida species, and Cryptococcus neoformans ; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 μM. At sub-MICs (<5 μM), BM2 chemosensitized to fluconazole (FLC) S. cerevisiae strains functionally hyperexpressing fungal lanosterol 14α-demethylase and resistance-conferring transporters of azole drugs. BM2 chemosensitized to FLC some FLC-resistant clinical isolates of C. albicans and C. dubliniensis and chemosensitized to itraconazole clinical isolates of C. krusei that are intrinsically resistant to FLC. The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells. BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.