Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy

Abstract

The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.

Figure 1. ML316 is a potent, highly selective fungicide

(a) Structures of ML316, original screen hit and inactive analog. Minimal inhibitory concentrations (MIC) are indicated for the C. albicans strain CaCi-2. For ML316 1 µM = 0.35 µg/ml. (b) Glucose-grown S. cerevisiae are not sensitive to ML316. Standard antifungal susceptibility testing was performed in YNB-CSM media with either glucose (Glu, 2% w/v) or glycerol (Gly, 2% w/v) as the carbon source. Optical densities were measured after 24 h at 30 °C and standardized to drug-free controls. The mean of three independent wells is shown. Error bars: s.e.m. (c) ML316 reduces growth of fluconazole-resistant C. albicans (strain CaCi-2), an effect that is increased by fluconazole (Flu). Assays in the presence or absence of Flu (8 µg/ml) were performed as in (b) except growth medium was RPMI supplemented with 2% glucose. The mean of two independent wells is shown. (d) ML316 inhibits growth of increasingly azole-resistant C. albicans strains isolated from the same HIV-infected patient over a 2-year interval. Strain designations are indicated to the left of the heat maps. Sensitivity testing was performed as in (c). The mean of results from two independent experiments is depicted. (e) ML316 is not cytotoxic to human cell lines under respiratory conditions. Viability was assessed by standard resazurin dye-reduction assay after 72 h growth at 37 °C in DMEM supplemented with galactose (10 mM) and dialyzed fetal bovine serum (10%). Data points depict the mean of measurements from 2 independent wells for each condition tested. (f) ML316 is fungicidal. A standard growth assay was performed as in (c) at either 30 or 37 °C. After 24 h, aliquots were spotted onto YPD agar plates and cultured for an additional 2 days. Scale bar = 10 mm. Data in (c), (e), and (f) are derived from one representative experiment. Two independent experiments yielding similar results were performed.

Figure 2. Genetic evidence for Mir1 as primary target of ML316

(a) ML316-sensitivity is correlated with MIR1 gene dosage and mutation status. Antifungal susceptibility testing with ML316 was performed using wild type and genetically modified C. albicans strains. Relative growth as monitored by OD₆₀₀ was normalized to untreated control for each strain and is displayed in heat map format. Data represent the mean of results from two independent experiments. (b and c) ML316 treatment phenocopies the deletion of MIR1 in S. cerevisiae. Change in optical density over time as a measure of growth was monitored in liquid cultures of wild type S. cerevisiae and the indicated mutant strains. Assays were performed in (b) glucose- or (c) galactose-supplemented synthetic media with vehicle (DMSO) or ML316 (500nM) added as indicated. Results are from one of two independent experiments. The mean of measurements from 4 independent wells is depicted.

Figure 3. Biochemical confirmation of ML316 as an inhibitor of Mir1

(a) ML316 inhibits phosphate-induced swelling and disruption of purified C. albicans mitochondria. The integrity of purified mitochondria isolated from strains expressing an additional copy of either the wild type (MIR1) or drug-resistant (mir1) gene version was monitored by optical density (OD). The effect of various inorganic anions on mitochondrial integrity in the presence or absence of ML316 (5 µM) is presented. Buffer alone served as baseline. Initial measurements were acquired approximately 10 seconds after addition of mitochondria to each reaction. Three independent aliquots of mitochondria were assayed for each condition. The entire experiment was performed twice. The mean and s.d. of measurements from one of these experiments is shown. (b) ML316 reduces the mitochondrial oxygen consumption (MOC) of C. albicans expressing an additional copy of MIR1 but not mir1R. The MOC rate of C. albicans strains grown in glycerol was measured 10 min after addition of ML316 (10 µM) or vehicle control (DMSO). Three independent aliquots of mitochondria were assayed in each of two independent experiments. The mean of one is shown. Error bars: s.e.m. * p<0.025 compared to DMSO-treated control (unpaired, two-tailed, parametric t-test).

Figure 4. ML316 renders respiration toxic and increases citrate levels

(a) C. albicans strain CaCi2 with genetic deletion of mitochondrial COX1 are resistant to ML316 but are unable to respire or grow in the presence of fluconazole. Serial dilutions of CaCi-2 or CaCi-2 cox1 were spotted on media with carbon sources supporting fermentation (glucose) or enforcing respiration (glycerol) in presence or absence of ML316 (5 µM) or Flu (16 µM) as indicated. Plates were imaged after 2 days at 30 °C. (b) Citrate levels increase dramatically upon treatment with ML316 but not with other mitochondrial poisons. LC/MS was used to measure levels of citrate in C. albicans grown in synthetic defined media (2% glucose) following 30 or 60 min exposure to ML316 (5 µM), Antimycin (1 µM), or 2,4-dinitrophenol (25 µg/mL). All values were normalized to the mean for DMSO-treated samples. Values represent the mean and s.e.m. of determinations from 2 independent experiments, each consisting of 3 independent cultures. (c) ML316 increases citrate levels in wild-type Candida but not in ML316-resistant mutant strains expressing either mir1R or carrying a deletion of COX4. Citrate levels were determined by LC/MS in strains growing in glucose after 60 min exposure to ML316 (5 µM). In the genotypes indicated on the x-axis, “V” denotes empty vector control. Citrate levels were normalized to the mean of wild type, DMSO-treated samples. Mean and s.e.m. from 2 independent experiments each consisting of 3 independent cultures are shown.

Figure 5. ML316 is active against azole-resistant C. albicans in mice

(a) Mice were treated with corticosteroids to induce susceptibility and infected sublingually with strain CaCi-2. Three days later, they were randomly assigned to treatment as indicated (n=8/ group except fluconazole (Flu) n=7). ML316 was added to drinking water (2.85 µM ML316/ 1% w/v sucrose) while Flu was administered intraperitoneally (10 mg/kg/day). Antifungal activity was assessed by measuring residual colony forming units (CFU) persisting on the tongue after completing 2 days of therapy. Mean and s.d. are displayed. The statistical significance of differences between treatment groups was determined by Mann Whitney test (unpaired, 2 tailed, non-parametric). P values are indicated above the relevant comparisons (Flu vs ML316; not significant). (b) Photomicrographs of PAS-stained tissue sections to assess fungal morphology are presented. Arrow heads indicate fungal foci which appear dark pink against the lighter staining of the keratinized papillae of the host tongue.