Antifungal activity in plants from Chinese traditional and folk medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: From over 100 Chinese clinical trial publications, we retrieved 22 commercial preparations and 17 clinical prescriptions used as Traditional Chinese Medicine (TCM) for treating mycotic vaginitis, typically caused by Candida albicans. The 8 most frequently used plants as well as another 7 TCM and 18 folk medicinal plants used in the South of China for antifungal therapy were investigated for in vitro antifungal activity. MATERIALS AND METHODS: For each plant we tested 4 extracts prepared with different solvents (water, ethanol, acetone, and n-hexane) for inhibition of Candida albicans and Saccharomyces cerevisiae growth in liquid culture. RESULTS: Some plants have quite strong antifungal activity, such as Tujinpi (Pseudolarix kaempferi Gord.), of which each extract could significantly inhibit the growth of both tested fungi. In addition, the acetone extract of Kushen (Sophora flavescens Ait.), the ethanol, acetone, and hexane extracts of Guanghuoxiang (Pogostemon cablin (Blanco) Benth.) and Gaoliangjiang (Alpinia officinarum Hance), the hexane extract of Dingxiang (Eugenia caryophyllata Thunb.), and the ethanol and acetone extracts of Kulianpi (Melia toosendan Sieb. et Zucc.) and Laliao (Polygonum hydropiper L.), all inhibited Candida albicans growth by more than 50%. In some cases growth inhibition was even comparable to that by the clinically used antifungal miconazole, which we used as our positive control. CONCLUSIONS: The majority of plants, whose clinical use for antifungal treatment is well supported within TCM or Chinese folk medicine, show in vitro antifungal activity against Candida albicans. Since Candida species represent the most common fungal pathogen of humans, these results provide more scientific evidence supporting the clinical application of these plants, and can serve as a starting point for new drug discovery from TCM and Chinese folk medicine.

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.

A fungicidal piperazine-1-carboxamidine induces mitochondrial fission-dependent apoptosis in yeast.

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.

Design and synthesis of a series of piperazine-1-carboxamidine derivatives with antifungal activity resulting from accumulation of endogenous reactive oxygen species.

In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure-activity relationship studies showed that piperazine-1-carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R(3) and R(5) positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine-1-carboxamidine derivatives to the accumulation of endogenous ROS.

Phosphorylation, lipid raft interaction and traffic of alpha-synuclein in a yeast model for Parkinson.

Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated alpha-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of alpha-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced alpha-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for alpha-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the alpha-synuclein membrane interaction. In addition, alpha-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of alpha-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.

Protein folding diseases and neurodegeneration: lessons learned from yeast.

Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism for studying fundamental cellular processes across the eukaryotic kingdom including man. In this respect, complementation assays, in which the yeast protein is replaced by a homologous protein from another organism, have been very instructive. A newer trend is to use the yeast cell factory as a toolbox to understand cellular processes controlled by proteins for which the yeast lacks functional counterparts. An increasing number of studies have indicated that S. cerevisiae is a suitable model system to decipher molecular mechanisms involved in a variety of neurodegenerative disorders caused by aberrant protein folding. Here we review the current knowledge gained by the use of so-called humanized yeasts in the field of Huntington's, Parkinson's and Alzheimer's diseases.

A yeast-based model of alpha-synucleinopathy identifies compounds with therapeutic potential.

We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.

Characterization of alpha-synuclein aggregation and synergistic toxicity with protein tau in yeast.

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of alpha-synuclein as well as the coexpression of protein tau. The data show that aggregation of alpha-synuclein is a nucleation-elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of alpha-synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of alpha-synuclein occurs via autophagy and proteasomes and that aggregation of alpha-synuclein disturbs endocytosis. Reminiscent of observations in double-transgenic mice, coexpression of alpha-synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of alpha-synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of alpha-synuclein and protein tau.