Niphimycin Research Articles

Enhancing Effects on Vacuole-Targeting Fungicidal Activity of Amphotericin B

Invasive fungal infections are major threats for immunocompromised patients as well as for those undergoing cancer chemotherapy. Amphotericin B (AmB), a classical antifungal drug with a polyene macrolide structure, is widely used for the control of serious fungal infections. However, the clinical use of this antifungal drug is limited by its side effects and the emergence of drug-resistant strains. AmB lethality has been generally attributed to alterations in plasma membrane ion permeability due to its specific binding to plasma membrane ergosterol. Recent studies with Saccharomyces cerevisiae and Candida albicans reveal the vacuole disruptive action as another cause of AmB lethality on the basis of marked amplification of its activity in combination with allicin, an allyl-sulfur compound from garlic. The enhancing effect of allicin is dependent on the inhibition of ergosterol-trafficking from the plasma membrane to the vacuole membrane, which is considered to be a cellular response to protect against disintegration of the vacuole membrane. The polyol macrolide niphimycin (NM) also possesses vacuole-targeting fungicidal activity, which is greater than that of AmB and nystatin. The alkyl side chain attached to the macrolide ring of NM is considered to possess an allicin-like inhibitory effect on the intracellular trafficking of ergosterol. The vacuole-targeting fungicidal activity was additionally detected with a bactericidal cyclic peptide polymyxin B (PMB), and was markedly enhanced when administered together with allicin, monensin, or salinomycin. The synergistic fungicidal activities of AmB and allicin may have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.

Enhancement effect of N-methyl-N″-dodecylguanidine on the vacuole-targeting fungicidal activity of amphotericin B against the pathogenic fungus Candida albicans

The alkylguanidium chain attached to the polyol lactone ring of niphimycin (NM) is considered a requisite for the fungicidal activity of NM characterized by vacuole membrane fragmentation and oxidative stress induction. The addition of N-methyl-N″-dodecylguanidine to the medium can enhance the vacuole-targeting fungicidal activity of amphotericin B (AmB), in which the lactone ring has no such alkylguanidium chain, on Saccharomyces cerevisiae cells. In this study, the enhancement effect of N-methyl-N″-dodecylguanidine on the vacuole-targeting fungicidal activity of AmB was examined against Candida albicans in RPMI 1640 medium at 37 °C. N-methyl-N″-dodecylguanidine was lethal to C. albicans cells and additionally enhanced the vacuole disruptive activity of AmB against this pathogenic fungus. N-methyl-N″-dodecylguanidine elevated the generation of cellular reactive oxygen species when added alone in a dose-dependent manner, but its enhancement effect on AmB lethality did not accompany amplification of oxidative stress induction. The fungal vacuoles were protected against the disruptive damage even if cells were treated with H(2)O(2) alone at a lethal concentration or treated with H(2)O(2) at a sublethal concentration in combination with AmB. N-methyl-N″-dodecylguanidine was ineffective in enhancing AmB lethality or AmB-induced vacuole disruption when cells had been pretreated with ergosterol. Ergosterol-dependent mechanism is thus considered to be a possible target of N-methyl-N″-dodecylguanidine in enhancing the vacuole-targeting fungicidal activity of AmB in C. albicans cells.

Synergistic Fungicidal Activities of Amphotericin B and N-Methyl-N″-dodecylguanidine: A Constituent of Polyol Macrolide Antibiotic Niphimycin

The synergy between the alkylguanidinium chain of niphimycin (NM), a polyol macrolide antibiotic, and polyene macrolide amphotericin B (AmB) without such an alkyl side chain was examined using N-methyl-N"-alkylguanidines as its synthetic analogs. Among the analogs, N-methyl-N"-dodecylguanidine (MC12) most strongly inhibited the growth of Saccharomyces cerevisiae cells and those of other fungal strains in synergy with AmB. MC12 itself was not lethal but the analog could be a cause of a rapid cell death progression of yeast cells in the presence of AmB at a nonlethal concentration. Their combined actions resulted in the generation of NM-like fungicidal activity that depended on plasma membrane disability and cellular reactive oxygen species production. We also found an aberrant vacuolar morphogenesis and an associated vacuolar membrane disability in cells treated simultaneously with MC12 and AmB, as in the case of NM-treated cells. These findings support the idea that the alkylguanidinium chain plays a major role in the fungicidal activity of NM in cooperation with the polyol lactone ring as its enhancer.

Structure–activity relationship studies on niphimycin, a guanidylpolyol macrolide antibiotic. Part 1: The role of the N-methyl-N″-alkylguanidinium moiety

Several N-methyl-N″-alkylguanidinium derivatives were synthesized and used as simplified analogues of niphimycin (NM), a guanidylpolyol macrolide, in structure–activity relationship studies. The C16-alkylated derivative exerted fungicidal activity by directly damaging the fungal plasma membrane and inducing oxidative stress in a manner similar to niphimycin. These results indicate that the N-methyl-N″-alkylguanidinium moiety is required for antifungal activity by NM.

Synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of polyol macrolide antibiotic niphimycin

The polyol macrolide niphimycin (NM) exhibited fungicidal activity against Saccharomyces cerevisiae cells accompanying the leakage of cytoplasmic components including nucleotide-like materials in addition to K + at 10 μM or above. Such a dynamic change in the plasma membrane was observed upon treatment of cells with H 2O 2 but not with the polyene macrolide antibiotic amphotericin B (AmB). The NM-induced cell death could be prevented by the exogenous addition of phosphatidylcholine (PC) whereas such a protective effect was only weakly observed with ergosterol, the molecular target of AmB. NM-treated cells were further characterized with a dramatic loss of glutathione even at a dose of 5 μM or less, representing NM-triggered metabolic conversion of the antioxidant molecule. NM-treatment indeed accelerated the cellular production of reactive oxygen species (ROS) such as H 2O 2 detectable with a specific fluorescent probe in a dose-dependent manner. These results suggested a synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of NM against S. cerevisiae.

Synergistic Combination of Direct Plasma Membrane Damage and Oxidative Stress as a Cause of Antifungal Activity of Polyol Macrolide Antibiotic Niphimycin

The polyol macrolide niphimycin (NM) exhibited fungicidal activity against Saccharomyces cerevisiae cells accompanying the leakage of cytoplasmic components including nucleotide-like materials in addition to K+ at 10 microM or above. Such a dynamic change in the plasma membrane was observed upon treatment of cells with H2O2 but not with the polyene macrolide antibiotic amphotericin B (AmB). The NM-induced cell death could be prevented by the exogenous addition of phosphatidylcholine (PC) whereas such a protective effect was only weakly observed with ergosterol, the molecular target of AmB. NM-treated cells were further characterized with a dramatic loss of glutathione even at a dose of 5 microM or less, representing NM-triggered metabolic conversion of the antioxidant molecule. NM-treatment indeed accelerated the cellular production of reactive oxygen species (ROS) such as H2O2 detectable with a specific fluorescent probe in a dose-dependent manner. These results suggested a synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of NM against S. cerevisiae.