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.