With more than 1.5 million deaths worldwide each year, invasive mycosis has become a serious global health issue. The limited supply of antifungal drugs, the emergence of drug-resistant species, and lack of rapid diagnostic tools hinder effective management of fungal infections. Our research focuses on exploring new chemical entities with novel mode of action, enhancing the currently available antifungal drugs, and discover new diagnostics tools.
To explore new antifungal agents, we use traditional screening methods to identify chemical entities with promising antifungal activities against various fungal pathogens such as Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In addition, we are interested in identifying compounds that interfere with the major virulence factors in fungi such as biofilm, yeast-hyphae transition, and proteolytic and lipolytic activities. We also are interested in identifying mechanisms of action of novel antifungal drugs such as auranofin, ebselen, dibromoquinoline [1-3].
Figure 1. Glutathione as a potential target of ebselen. (A and B) The percent growth of yeast cells (OD600 after 24 h) incubated with ebselen (2μg/ml) in YPD broth was determined in relation to the DMSO treatment. (C) Saturated cultures of yeast cells were diluted to 1:5 and grown for 2.5 h. The cells were sonicated, and the amount of glutathione was determined using a glutathione assay kit. The absorbance measured using a spectrophotometer indicates the glutathione production in each strain. (D) Yeast cells were treated with ebselen (20μg/ml) for 2.5 h and the glutathione concentration was measured as indicated above. The results are expressed as percent glutathione production relative to untreated control groups. Statistical analysis was calculated using the two-tailed Student's t-test. P values of *P≤0.05 and **P≤0.01 are considered as significant.
Figure 2. Auranofin targets mitochondrial protein(s). (A) Chemogenomic profiling of S. cerevisiae with treatment of auranofin. The strain abundance was normalized using EdgeR and shown. (B) Growth curve of wild type (BY4743) and heterozygous deletion strains (mia40∆, acn9∆, coa4∆, rad18∆, and nsi1∆) in the presence of indicated concentration of auranofin in YPD broth were determined. (C) The percent growth of yeast cells (OD600 after 24 h) incubated with auranofin (6.25 μg/mL) in YPD broth was determined in relation to the DMSO treatment. The results are presented as means ± SD (n = 3). Statistical analysis was calculated using the two-tailed Student’s t-test. P-values (*P ≤ 0.05) (**P ≤ 0.01) are considered as significant. (D) Yeast cells grown in YPD broth overnight were serially diluted and spotted on solid YPD agar containing auranofin (6.25 μg/mL) or DMSO and the CFU were shown. (E) Comparison of Lee et al. (2014) HIP results with our 85 strains are shown as a Venn diagram.
Using a drug repurposing approach, we were able to identify several agents with potent azole chemosensitizing activities. We reported a potent synergistic relationship between sulfa drugs and azoles especially against Candida albicans. Competition assays indicated para-aminobenzoic acid (PABA) was able to eliminate the synergistic relationship between sulfa drugs and azoles which suggests the mode of action of sulfa drugs in these combinations is mediated through the interference with the folate biosynthesis . Another class of compounds that interacted synergistically with azole drugs was trans-stilbene derivatives . Of these compounds, Ospemifene, an estrogen receptor modulating compound, exhibited promising broad-spectrum synergistic activities with itraconazole and was effective against multiple fungal pathogens such as Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus. Our data indicate that ospemifene interferes with several efflux transporters such as Mdr1p and Cdr1p, and thus enhances the antifungal activity of azoles by increasing their intracellular levels.
Figure 3. Effect of the ospemifene-itraconazole combination on the growth kinetics of different fungal
species. Overnight cultures of fungal isolates were diluted to 0.5–2.5 × 103 CFU/ml in RPMI 1640 medium. Cells were treated with ospemifene, itraconazole (ITC), or a combination of the two drugs at the indicated concentrations. Cells were incubated at 35 °C for 48–72 h, and OD595 values were measured at different time points (0, 6, 12, 18, 24, 36 and 48 h). *Indicates statistical significance relative to the treated control while (#) indicates statistical significance (P < 0.05) relative to individual treatments with ospemifene or itraconazole. The statistical significance was determined by multiple t-tests using the Holm-Sidak statistical method for multiple comparisons.
Candida albicans are the single most prevalent cause of fungal bloodstream infections worldwide causing significant mortality as high as 50 percent. This high mortality rate is, in part, due to the inability to initiate an effective antifungal therapy early in the disease process. Mortality rates significantly increase after 12 hours of delay in initiating the appropriate antifungal therapy following a positive blood culture. In collaboration with Dr. Ji-Xin Cheng, we are exploring the stimulated Raman scattering (SRS) imaging to probe for metabolic differences between fluconazole-susceptible and -resistant strains at a single cell level in search of a metabolic signature. C–H frequency (2850 cm–1) SRS imaging revealed a substantial difference in lipogenesis between the fluconazole-susceptible and -resistant C. albicans. Exposure to fluconazole, an antimicrobial drug that targets ergosterol biosynthesis, only affected the lipogenesis in the susceptible strain. These results show that single-cell metabolic imaging via SRS microscopy can be used for the rapid detection of azole-resistant species .
Figure 4. Fluconazole exposure has contrasting effects on metabolism in fluconazole-susceptible and -resistant C. albicans strains. (a) Exposure to fluconazole downregulates 2NBDG (glucose analog) uptake in fluconazole-susceptible strain, while it upregulates 2NBDG uptake in the fluconazole- resistant strain. (b) Quantification of 2NBDG uptake alteration induced by fluconazole treatment in fluconazole-susceptible and -resistant C. albicans strains. (c) C−H frequency SRS imaging reveals exposure to fluconazole attenuates lipid accumulation in the susceptible strain and no significant change in the resistant strain. (d) Quantification of lipid accumulation alteration induced by fluconazole treatment in fluconazole-susceptible and -resistant C. albicans strains. (e) Visualization of de novo lipogenesis via C−D frequency SRS imaging reveals de novo lipogenesis is attenuated by fluconazole treatment in the susceptible strain; on the contrary, the lipogenesis rate is relatively the same in the absence and presence of fluconazole in the resistant strain. (f) Quantification of de novo lipogenesis alteration induced by fluconazole treatment in both fluconazole-susceptible and -resistant C. albicans strains. Student’s t test used for statistical analysis. ***p < 0.001; ns (not significant).
1. Thangamani, S., et al., Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway. Front Cell Infect Microbiol, 2017. 7: p. 4.
2. Thangamani, S., et al., Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells. Biochim Biophys Acta Gen Subj, 2017. 1861(1 Pt A): p. 3002-3010.
3. Mohammad, H., et al., Discovery of a Novel Dibromoquinoline Compound Exhibiting Potent Antifungal and Antivirulence Activity That Targets Metal Ion Homeostasis. ACS Infect Dis, 2018. 4(3): p. 403-414.
4. Eldesouky, H.E., et al., Reversal of Azole Resistance in Candida albicans by Sulfa Antibacterial Drugs. Antimicrob Agents Chemother, 2018. 62(3).
5. Eldesouky, H.E., et al., Ospemifene displays broad-spectrum synergistic interactions with itraconazole through potent interference with fungal efflux activities. Sci Rep, 2020. 10(1): p. 6089.
6. Karanja, C.W., et al., Stimulated Raman Imaging Reveals Aberrant Lipogenesis as a Metabolic Marker for Azole-Resistant Candida albicans.Anal Chem, 2017. 89(18): p. 9822-9829.
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