Surface-active fungicidal D-peptide inhibitors of the plasma membrane proton pump that block azole resistance.
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 microM. At sub-MICs (<5 microM), BM2 chemosensitized to fluconazole (FLC) S. cerevisiae strains functionally hyperexpressing fungal lanosterol 14alpha-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.
Monk
Brian C
BC
Molecular Microbiology Laboratory, Department of Oral Sciences, School of Dentistry, University of Otago, P.O. Box 647, Dunedin 9001, New Zealand. brian.monk@stonebow.otago.ac.nz
Niimi
Kyoko
K
Lin
Susan
S
Knight
Allison
A
Kardos
Thomas B
TB
Cannon
Richard D
RD
Parshot
Rekha
R
King
Amanda
A
Lun
David
D
Harding
David R K
DR
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D002175:Candida / Q000145:classification / Q000187:drug effects; D002176:Candida albicans / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects*; D003455:Cryptococcus neoformans / Q000187:drug effects; D025141:Drug Resistance, Fungal; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D019151:Peptide Library; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors*
D000935:Antifungal Agents; D001393:Azoles; D009842:Oligopeptides; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins; C496527:arginyl-arginyl-arginyl-phenylalanyl-tryptophyl-tryptophyl-phenylalanyl-arginyl-aginyl-argininamide; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases