Novel fungicidal benzylsulfanyl-phenylguanidines.

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ≤ 10 µM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC=50-100 µM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ≤ 121 µM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC=5 µM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ≤ 4 µM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.

Antifungal carbazoles.

Carbazole derivatives are well known for their various pharmacological activities, including anti-HIV, anticancer, antibacterial and antifungal activities. This review will focus on carbazoles that possess antifungal activity against Candida albicans, the major human fungal pathogen. In our search for new fungicidal compounds, we identified a series of substituted carbazoles, termed N-alkylated 3,6-dihalogenocarbazoles, that exhibit fungicidal activity against C. albicans and the emerging pathogen Candida glabrata. The most potent fungicidal compounds of this series were characterized by minimal fungicidal concentration (MFC) between 8.5 and 25 microM. To analyse the structural determinants for fungicidal activity of these carbazole derivatives, we selected 10 such derivatives and performed further analyses. Interestingly, some of these N-alkaylated 3,6-dihalogenocarbazoles were active against Candida biofilms grown in microtiterplates. In this review, we will further discuss the putative therapeutic potential of the antifungal carbazole compounds as antimycotics.

Fungicidal activity of truncated analogues of dihydrosphingosine.

The minimal fungicidal concentration (MFC) of dihydrosphingosine (DHS), phytosphingosine (PHS), and five short-chain DHS derivatives was determined for Candida albicans and Candida glabrata. In this respect, a C15- and a C17-homologue of DHS showed a 2- to 10-fold decreased MFC as compared to native DHS (i.e. C18-DHS). DHS derivatives that were active, that is, comprising 12, 15, 17, or 18 carbon atoms, induced accumulation of reactive oxygen species (ROS) in C. albicans.

SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis.

The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)(2)C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)(2)C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in beta-1,6-glucan biosynthesis. We demonstrate that a Deltaskn1 mutant lacks M(IP)(2)C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)(2)C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae.

DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae.

DmAMP1, an antifungal plant defensin from Dahlia merckii, was shown previously to require the presence of sphingolipids for fungicidal action against Saccharomyces cerevisiae. Sphingolipids may stabilize glycosylphosphatidylinositol (GPI)-anchored proteins, which interact with DmAMP1, or they may directly serve as DmAMP1 binding sites. In the present study, we demonstrate that S. cerevisiae disruptants in GPI-anchored proteins showed small or no increased resistance towards DmAMP1 indicating no involvement of these proteins in DmAMP1 action. Further, studies using an enzyme-linked immunosorbent assay (ELISA)-based binding assay revealed that DmAMP1 interacts directly with sphingolipids isolated from S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol. Therefore, DmAMP1 antifungal action involving membrane interaction with sphingolipids and ergosterol is proposed.

The antifungal activity of RsAFP2, a plant defensin from raphanus sativus, involves the induction of reactive oxygen species in Candida albicans.

RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant DeltagcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.

Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast.

The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.