Discovery of Natural Products With Antifungal Potential Through Combinatorial Synergy

Abstract

The growing prevalence of antifungal drug resistance coupled with the slow development of new, acceptable drugs and fungicides has raised interest in natural products (NPs) for their therapeutic potential and level of acceptability. However, a number of well-studied NPs are considered promiscuous molecules. In this study, the advantages of drug-drug synergy were exploited for the discovery of pairwise NP combinations with potentiated antifungal activity and, potentially, increased target specificity. A rational approach informed by previously known mechanisms of action of selected NPs did not yield novel antifungal synergies. In contrast, a high-throughput screening approach with yeast revealed 34 potential synergies from 800 combinations of a diverse NP library with four selected NPs of interest (eugenol, EUG; β-escin, ESC; curcumin, CUR; berberine hydrochloride, BER). Dedicated assays validated the most promising synergies, namely, EUG + BER, CUR + sclareol, and BER + pterostilbene (PTE) [fractional inhibitory concentrations (FIC) indices ≤ 0.5 in all cases], reduced to as low as 35 (BER) and 7.9 mg L^-1 (PTE). These three combinations synergistically inhibited a range of fungi, including human or crop pathogens Candida albicans, Aspergillus fumigatus, Zymoseptoria tritici, and Botrytis cinerea, with synergy also against azole-resistant isolates and biofilms. Further investigation indicated roles for mitochondrial membrane depolarization and reactive oxygen species (ROS) formation in the synergistic mechanism of EUG + BER action. This study establishes proof-of-principle for utilizing high-throughput screening of pairwise NP interactions as a tool to find novel antifungal synergies. Such NP synergies, with the potential also for improved specificity, may help in the management of fungal pathogens.

Keywords: Saccharomyces cerevisiae; Zygosaccharomyces bailii; crop pathogens; drug combinations; fungal pathogens; fungicide combinations.

FIGURE 1

Checkerboard assays of combinatorial growth effects of eugenol, β-escin, and curcumin in S. cerevisiae. Assays were performed according to the EUCAST procedure in YPD broth with S. cerevisiae W303 at the indicated concentrations of eugenol, β-escin, and curcumin. Growth values (scale to the right) represent means from three independent experiments, calculated as percentages of growth (OD₆₀₀) with the NPs relative to the minus-NP control. FICI, fractional inhibitory concentration index, calculated from data after 24 h growth at 30°C; growth values < 5% were assigned as no-growth (Hsieh et al., 1993). Corresponding data for S. cerevisiae BY4743 are shown in Supplementary Figure 1.

FIGURE 2

Effect of metabolic environment on the stability of combinatorial interactions. (A) Growth curves for S. cerevisiae W303 in YEP medium supplemented with either glucose, glycerol, or ethanol (all at 2% w/v). Each point represents the mean of three independent experiments ± SEM (error bars did not exceed the dimensions of the symbols). (B) S. cerevisiae W303 was treated in checkerboard format with combinations of eugenol, β-escin, and curcumin in YEP supplemented with 2% either glucose (black), glycerol (pink), or ethanol (green). Percentage growth was used to calculate FIC indices. Each bar represents the mean of three independent experiments ± SEM. The corresponding checkerboard data are presented in Supplementary Figure 2.

FIGURE 3

A screen of library NPs in combinations with selected NPs of interest, against the growth of S. cerevisiae. Left: normalized growth of S. cerevisiae W303 was calculated from OD₆₀₀ values after 24 h with and without NPs, for each of the library NPs both in the absence (x-axis) or in the presence (y-axis) of 750 μM eugenol (EUG), 12.5 μM β-escin (ESC), 50 μM curcumin (CUR), or 350 μM berberine (BER). Each point represents the mean ± SEM calculated from two independent experiments. Right: effect strengths were determined from [(% growth with library agent) - (% growth with library agent + second agent)] for the different combinations; color is used to highlight those with an effect strength > 50. The underlying data for each combination from the screen are listed in Supplementary Table 1.

FIGURE 4

Corroboration of synergies in S. cerevisiae. (A) Mean effect strengths ± SEM from each screen (Figure 3) for selected combinations of interest (n = 2). (B) Checkerboard assays of combinatorial growth effects performed according to the EUCAST procedure in YPD broth with S. cerevisiae W303 at the indicated concentrations of eugenol, curcumin, berberine, sclareol, and pterostilbene. The growth values represent the mean of three independent experiments calculated as percentages of growth (OD₆₀₀) with the natural products relative to the minus-NP control, after 24 h growth at 30°C. FICI, fractional inhibitory concentration index, calculated from the data and where growth < 5% of the control was assigned as no-growth (Hsieh et al., 1993). (C) Chemical structures of library compounds giving the strongest effect strengths from each screen and validated in panel (B). Checkerboard data for additional combinations are presented in Supplementary Figure 4.

FIGURE 5

Mitochondrial membrane depolarization in eugenol- and berberine-treated yeast cells. (A) Checkerboard assays of combinatorial growth effects were performed as described in Figures 1, 4. Combination concentrations that were subsequently tested for mitochondrial membrane depolarization are circled. (B) Microscopic imaging of S. cerevisiae cells treated with or without 94 μM EUG and 188 μM BER for 24 h, stained with rhodamine-123. Images were captured using a ×40 objective lens, through a FITC-filter; scale bar, 20 μm. Images are representative of three biological replicates. (C) Flow cytometric histograms for cells incubated for 24 h without (control) or with the indicated concentrations of EUG and BER. Cells were then stained with rhodamine 123 before analysis of fluorescence; a.u., arbitrary units. (D) Observed effects of combinations were obtained experimentally from median fluorescence of rhodamine 123-stained cells exposed to the EUG + BER combination [derived from corresponding flow cytometric data as in panel (B)], normalized to the no drug control (100%). Expected effects were calculated by multiplication of the % median-fluorescence determinations obtained for the corresponding individual-compound effects. Values represent means ± SEM from four independent experiments: *p < 0.05 and **p < 0.01 according to paired t-tests. EUG, eugenol; BER, berberine.

FIGURE 6

Effect of antioxidants on the eugenol plus berberine synergy. Values plotted to represent mean FICI values from three independent checkerboard experiments performed with S. cerevisiae W303, as described in Figures 1, 4, with the inclusion of the antioxidants at the specified concentrations. Bar heights show means ± SEM. *p < 0.05 and **p < 0.05, according to unpaired t-tests. The relevant checkerboard data are presented in Supplementary Figure 6.

FIGURE 7

Eugenol plus berberine synergy in deletion strains defective for mitochondrial antioxidant proteins. (A) Checkerboard assays of combinatorial growth effects performed according to the EUCAST procedure in YPD broth within S. cerevisiae BY4743 (WT) and isogenic deletion mutants Δsod2, Δccp1, and Δogg1 at the indicated concentrations of eugenol and berberine. The growth values represent the mean of three independent experiments calculated as percentages of growth (OD₆₀₀) with the natural products relative to the minus-NP control, after 24 h at 30°C. FICI, fractional inhibitory concentration index, calculated from the data and where growth < 5% of the control was assigned as no-growth (Hsieh et al., 1993). (B) FIC indices are plotted from three independent checkerboard experiments, with bar-height showing mean ± SEM. **p < 0.01, unpaired t-test. ns, not significant.