Selective Metal Chelation by a Thiosemicarbazone Derivative Interferes with Mitochondrial Respiration and Ribosome Biogenesis in Candida albicans

ABSTRACT Metal chelation is generally considered as a promising antifungal approach but its specific mechanisms are unclear. Here, we identify 13 thiosemicarbazone derivatives that exert broad-spectrum antifungal activity with potency comparable or superior to that of fluconazole in vitro by screening a small compound library comprising 89 thiosemicarbazone derivatives as iron chelators. Among the hits, 19ak exhibits minimal cytotoxicity and potent activity against either azole-sensitive or azole-resistant fungal pathogens. Mechanism investigations reveal that 19ak inhibits mitochondrial respiration mainly by retarding mitochondrial respiratory chain complex I activity through iron chelation, and further reduces mitochondrial membrane potential and ATP synthesis in Candida albicans. In addition, 19ak inhibits fungal ribosome biogenesis mainly by disrupting intracellular zinc homeostasis. 19ak also stimulates the activities of antioxidant enzymes and decreases reactive oxygen species formation in C. albicans, resulting in an increase in detrimental intracellular reductive stress. However, 19ak has minor effects on mammalian cells in depleting intracellular iron and zinc. Moreover, 19ak exhibits low capacity to induce drug resistance and in vivo efficacy in a Galleria mellonella infection model. These findings uncover retarded fungal mitochondrial respiration and ribosome biogenesis as downstream effects of disruption of iron and zinc homeostasis in C. albicans and provide a basis for the thiosemicarbazone 19ak in antifungal application. IMPORTANCE The increasing incidence of fungal infections and resistance to existing antifungals call for the development of broad-spectrum antifungals with novel mechanisms of action. In this study, we demonstrate that a thiosemicarbazone derivative 19ak selectively inhibits mitochondrial respiration mainly by retarding mitochondrial respiratory chain complex I activity through iron chelation and inhibits ribosome biogenesis mainly by disrupting intracellular zinc homeostasis in C. albicans. In addition, 19ak exhibits low capacity to induce fungal resistance, minimal cytotoxicity, and in vivo antifungal efficacy. This study provides the basis of thiosemicarbazone derivative 19ak as a metal chelator for the treatment of fungal infections.


SUPPLEMENTARY EXPERIMENTAL METHODS.
Compound library. The thiosemicarbazone derivatives Triapine and COTI-2 were purchased from Med Chem Express (MCE). The other 87 thiosemicarbazones used in this study were previously synthesized by our collaborators (1,2). Their structures are shown in Table 1 and Table S1 to help the reader clearly understand the relationship between structure and activity. The effects of the compounds on cell proliferation were detected by measuring the absorbance at 570 nm in a microplate reader (BioTek, VT, USA). Three independent experiments were conducted.

Rescue effect of exogenous metal ions on fungal growth inhibition by 19ak.
Since the effects of thiosemicarbazone may be due to binding of intracellular metal ions, we investigated the impact of adding metal ions to the medium on the inhibitory effect of 19ak by performing antifungal susceptibility testing as described above in the presence of 50 μM, 100 μM, and 200 μM ZnSO4, FeSO4, and FeCl3.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) enrichment analysis. The Cluster Profiler R package was used for Gene Ontology (GO) enrichment analysis of the DEGs, with P-value < 0.05 considered significantly enriched (6). We also analyzed KEGG pathways that were significantly enriched in DEGs with the aim of understanding the advanced functions and roles of biological systems based on large, high-throughput molecular data sets (7). Mitochondrial complex activity was expressed as nmol/min/mg protein.

Analysis of mtΔψ.
Rh123 is a fluorescent stain that responds directly to mtΔψ. C. instructions. An overnight culture of C. albicans SC5314 was adjusted to 1 × 10 6 cells/mL with RPMI1640 medium and treated as described above for SOD activity.
The decomposition of H2O2 is directly accompanied by a decrease in absorbance at 240 nm, and the difference in absorbance per unit time (ΔA240) is a measure of catalase activity.
Quantitative real-time polymerase chain reaction (qPCR) analysis. C.
albicans SC5314 cells (1 × 10 6 cells/mL) were incubated with or without 19ak (0.5, 2 μg/mL) in RPMI1640 medium for 6 h. Total RNA was extracted using the hot phenol method, and the RNA quality was analyzed using a spectrophotometer (Eppendorf, Hamburg, Germany). The RNA was converted to cDNA using a cDNA Reverse Transcription kit (Accurate Biotechnology, Hunan, China). PCR detection based on SYBR Green was performed on an Eppendorf real-time PCR system (Eppendorf, Hamburg, Germany). The primer sequences used in this study are shown in Table S4.
Drug resistance study. Drug resistance studies were carried out by repeatedly treating C. albicans with an antifungal agent followed by MIC determination using the broth microdilution assay (9). Specifically, C. albicans cells incubated at 0.5 × MIC were adjusted to 1 × 10 3 cells/mL using RPMI1640 medium for the next passage of MIC measurement. This process was repeated 40 times.
Statistical analysis. One-way ANOVA test or Student's t-test (two-tailed, unequal variance) was used to assess the statistical significance of the difference between treatment group and control group. The Mantel-Cox test was used to compare differences between groups in survival analysis. Data were showed as mean ± standard deviation (SD). Statistical significance was determined according to the P value. *P < 0.05, **P < 0.01, ***P < 0.001.