A Fungal-Selective Cytochrome bc1 Inhibitor Impairs Virulence and Prevents the Evolution of Drug Resistance

Abstract

To cause disease, a microbial pathogen must adapt to the challenges of its host environment. The leading fungal pathogen Candida albicans colonizes nutrient-poor bodily niches, withstands attack from the immune system, and tolerates treatment with azole antifungals, often evolving resistance. To discover agents that block these adaptive strategies, we screened 300,000 compounds for inhibition of azole tolerance in a drug-resistant Candida isolate. We identified a novel indazole derivative that converts azoles from fungistatic to fungicidal drugs by selective inhibition of mitochondrial cytochrome bc₁. We synthesized 103 analogs to optimize potency (half maximal inhibitory concentration 0.4 ?M) and fungal selectivity (28-fold over human). In addition to reducing azole resistance, targeting cytochrome bc₁ prevents C. albicans from adapting to the nutrient-deprived macrophage phagosome and greatly curtails its virulence in mice. Inhibiting mitochondrial respiration and restricting metabolic flexibility with this synthetically tractable chemotype provides an attractive therapeutic strategy to limit both fungal virulence and drug resistance.

Figure 1. Discovery of Inz-1 by high-throughput phenotypic screening

A) Schematic of the high throughput screen for inhibitors of azole tolerance. B) Triage of hits from the screen, indicating number of compounds that passed each filter. C) Profiling of fungal growth under stress conditions in the presence of Inz-1 and other small molecules. Strain SC5314 was grown for 24hr in the presence of the indicated stressors and small molecule drugs (concentrations indicated in Experimental Procedures). Growth was measured by reading OD600 after 24hr. Results were normalized to the growth under each stressor condition (rows) with addition of DMSO used as negative control and visualized as a heatmap using TreeView. D) Effect of Inz-1 on growth of S. cerevisiae (strain BY4741) and C. albicans (SC5314) in media containing glucose or glycerol as carbon source. Figures in C-D depict data pooled from two independent experiments.

Figure 2. Inz-1 is a fungal-selective inhibitor of mitochondrial cytochrome bc1

A) Mutations found in cytochrome B (COB) in two Inz-1-resistant isolates, visualized by the Integrative Genomics Viewer (IGV). B) Inhibition of yeast cytochrome B enzymatic activity by Inz-1 in both wild-type and F90Y mutant mitochondria. C) Resistance of F90Y mutant to growth inhibition by Inz-1. D) Fluconazole tolerance of C. albicans CaCi-2 WT, treated with Inz-1, or rip1Δ/Δ deletion mutant. Growth at the indicated concentrations of fluconazole was measured by broth microdilution using OD600 as endpoint. E) Comparison of inhibition of cytochrome B activity in C. albicans and human (HEK293) mitochondria. F) Comparison of the effect of Inz-1 on proliferation of HepG2 and NIH-3T3 cells under respiring conditions (DMEM-galactose) with effect on proliferation of C. albicans in presence of 8 μg/mL fluconazole. Graphs in B-F each represent data pooled from two independent experiments, with error bars depicting S.E.M.

Figure 3. Structural analysis of Inz-1 binding to cytochrome bc1

A) Minimal Inhibitory Concentration (MIC) of Inz-1 for indicated cytochrome B mutants under respiratory growth conditions in S. cerevisiae. MIC’s were concordant from two independent experiments. B) Structure of yeast cytochrome B complex (PDB:1EZV) indicating location of Qi and Qo pockets. C) Structural alignment of yeast (PDB:1P84) and bovine (PDB:1SQV) cytochrome B showing Qo site and indicating location of two mutations that confer resistance to Inz-1. D) Computational docking of Inz-1 into Qo pocket of yeast cytochrome bc1 (PDB:1EZV) with location of resistance-conferring mutations overlaid.

Figure 4. Chemical optimization of Inz-1

104 analogs of Inz-1 were synthesized and tested for inhibition of yeast and human cytochrome B. Data for selected analogs are shown; results for the remaining compounds are shown in Table S1. Each data point represents the average of at least 2 independent experiments. Experiments were performed as described in Figure 2. Candida growth inhibition indicates the IC50 for growth inhibition of CaCi-2 by compounds in the presence of 8 μg/mL fluconazole. Selectivity was calculated by dividing growth inhibition IC50 of HepG2 cells by IC50 for growth inhibition of C. albicans.

Figure 5. Inz-5 renders fluconazole fungicidal and reduces the emergence of resistance

A) 2×10³ C. albicans wild-type (SC5314) cells were plated on media containing 64 mg/L fluconazole (top right), 10μm Inz-5 (bottom left) or both compounds in combination. Images were acquired after 4d incubation at 37C. B) 5×10³ C. albicans SC5314 cells were incubated in the presence of 32 mg/L fluconazole, 10 μM Inz-5, or both compounds while C. albicans rip1Δ/Δ were incubated in the presence and absence of 32 mg/L fluconazole. To measure clonogenic survival, dilutions were plated on YPD after 24 and 48 hr. Data are pooled from two independent experiments; error bars indicate S.E.M.

Figure 6. Inhibition of cytochrome bc1 by Inz-5 prevents adaptation to certain carbon sources

A) Growth of ~10⁴ C. albicans SC5314 cells on agar plates containing various carbon sources in the presence of a filter disc containing DMSO (top) or 10μg Inz-5 dissolved in DMSO (bottom). B) Co-culture of BFP-labeled C. albicans and GFP-tubulin-labeled mouse bone-marrow-derived macrophages. C) Quantification of effects on filamentation observed in (B); data from two independent experiments; error bars indicate S.E.M. D) Relative fungal survival quantified by resazurin dye reduction after 14 hr co-culture at MOI of 0.125 in the presence of mouse bone marrow-derived macrophages. Data from two independent experiments; error bars indicate S.E.M.

Figure 7. Genetic disruption of cytochrome bc1 severely reduces fungal virulence and fluconazole tolerance, but increases brain colonization

A) Kaplan-Meier analysis of survival after tail-vein infection of Balb/C mice with 5×10⁵ fungal cells of indicated strains. N = 7 mice per strain, data pooled from two independent experiments. B) Viable fungal burden isolated from brain 4d after infection with 6×10⁴ CFU of indicated strains. N = 6-7 mice per strain; data pooled from two independent experiments. C) Kaplan-Meier analysis of survival for mice infected with 10⁶ CFU of CaCi-2 then treated with fluconazole (24 mg/kg IP) once daily X 3d. N = 6-10 mice per strain; data pooled from two independent experiments. D) Mice were infected with 6×10⁴ CFU of the indicated strains, then treated with fluconazole (24 mg/kg IP, once daily X3). After treatment mice were monitored for 4 more days, then sacrificed and fungal burden in kidney and brain quantified. Data are pooled from two independent experiments.