https://orcid.org/0000-0003-2367-0915
Abstract
The rise in fungal infections caused by multidrug-resistant pathogens like Candida haemulonii sensu stricto presents a significant global health challenge. The common resistance to current treatments underscores the urgency to explore alternative therapeutic strategies, including drug repurposing.
To assess the potential of repurposing tafenoquine, an antimalarial agent, for antifungal use against C. haemulonii sensu stricto.
The efficacy of tafenoquine was tested using in vitro assays for minimum inhibitory concentration (MIC), minimum fungicidal concentration, biofilm inhibition, cell damage, cell membrane integrity, nucleotide leakage, sorbitol protection assay, and efflux pump inhibition. The compound’s cytotoxicity was assessed through a haemolysis assay, and in vivo safety and efficacy were tested using Tenebrio molitor larvae.
Tafenoquine exhibited potent fungicidal activity against C. haemulonii sensu stricto with an MIC of 4 mg/L and significantly inhibited biofilm formation by 60.63%. Tafenoquine also impaired mitochondrial functionality, leading to compromised cellular respiration. Despite these effects, tafenoquine did not cause significant protein leakage, indicating a distinct mechanism from membrane-targeting agents. In vivo study confirmed tafenoquine's non-toxic profile with no observed haemolysis or acute toxicity in the T. molitor model. During antifungal treatment with tafenoquine, a survival rate of approximately 60% was observed after 3 days.
The findings of this study highlight tafenoquine's potential as a promising candidate for antifungal drug repurposing, especially against C. haemulonii sensu stricto. Its effectiveness in inhibiting fungal growth and biofilm formation underscores its viability for further clinical development as a novel antifungal therapy.
Introduction
Candidemia is one of the most significant causes of morbidity and mortality in healthcare environments, particularly in intensive care units and among immunocompromised patients, such as those undergoing chemotherapy, organ transplants, or with complex medical conditions.1–3 This invasive fungal infection (IFI) not only increases hospital stays but also contributes to high healthcare costs due to the complexity of its management and the need for prolonged treatments.4
In recent years, there has been a notable rise in the incidence of IFIs caused by cryptic Candida species, which are often more difficult to identify and treat due to their diverse virulence factors and resistance mechanisms.5,6 Among these, the Candida haemulonii species complex has gained particular attention. In 2012, this group was redefined to include C. haemulonii sensu stricto, Candida duobushaemulonii, and C. haemulonii var. vulnera.3
The identification and treatment of infections caused by this complex are made more challenging by the organisms’ multidrug-resistant (MDR) characteristics.7 These yeasts often exhibit high minimum inhibitory concentrations (MICs) for fluconazole,8 a first-line antifungal therapy in many regions, particularly in low-income countries where alternative treatments may be limited. They also show reduced susceptibility to amphotericin B,9 a potent antifungal for severe infections. While echinocandins are generally effective, emerging echinocandin-resistant strains are a growing concern.10
The challenges of MDR fungal infections and limitations of existing antifungals make drug repurposing a promising strategy for new treatments.11 This strategy allows for the rapid identification of new therapeutic uses for existing drugs, bypassing the lengthy process of drug discovery and benefiting from known safety profiles.11–13 Recent studies have highlighted the potential of repurposing antimalarial drugs as antifungal agents,14 with tafenoquine—originally developed to treat malaria—emerging as a particularly strong candidate.15,16 This study explores tafenoquine’s potential as a novel antifungal against C. haemulonii sensu stricto, using both in vitro and in vivo approaches.
Materials and methods
Strain and culture conditions
C. haemulonii sensu stricto strain (132/23) used in this study was sourced from the library of the Center for Studies in Applied Medical Mycology at the Health Sciences Research Laboratory of the Universidade Federal of Grande Dourados. This strain was previously characterized for biofilm production and identified by sequencing (ITS) region of rDNA.17 The isolate, obtained from a blood culture, demonstrated resistance to amphotericin B and fluconazole.18 The strain was maintained in 20% glycerol at −80°C until use. For the experiments, it was cultured in Sabouraud agar (SDA) and incubated at 37°C for 48 h.
Minimum inhibitory concentration
The MIC was determined by broth microdilution following EUCAST guidelines.19 The sensitivity profile of the compound tafenoquine was assessed using dilutions ranging from 0.25 to 128 mg/L. Fungal suspensions were diluted in sterile distilled water and plated in 96-well plates at 2.5 × 105 cells/mL. The plates were incubated at 37°C for 48 h. Antifungal activity was determined spectrophotometrically at 530 nm and the MIC corresponded to the smallest compound concentrations able to inhibit 90% of growth. To determine the minimum fungicidal concentrations (MFC), 10 μL of each well from the MIC plates was transferred to SDA plates and incubated at 37°C for 48 h. Amphotericin B concentrations ranging from 0.03 to 16 mg/L were used as a resistance control. The procedure was conducted in duplicate.
Checkboard assay
Tafenoquine was evaluated combined with fluconazole, amphotericin B, and micafungin using the checkerboard microdilution method.20 Tafenoquine concentrations ranged from 0.25 to 128 mg/L, fluconazole from 0.125 to 64 mg/L, amphotericin B from 0.031 to 16 mg/L, and micafungin from 0.015 to 8 mg/L. For plate setup, 50 μL of tafenoquine was placed horizontally, and 50 μL of antifungal was placed vertically in a 96-well plate with a flat bottom. Subsequently, 100 μL of inoculum (105 cells/mL) was added to each well containing the compounds and eight wells containing only 2 × RPMI medium supplemented with 2% glucose and 165 mM MOPS (pH 7.0) (positive control). Sterility control was also performed, with eight wells containing only 2 × RPMI medium supplemented with 2% glucose and 165 mM MOPS (pH 7.0). The properly capped plates were incubated for 48 h at 37°C. The procedure was conducted in duplicate.
The interaction between the compounds was quantified by the Fractional Inhibitory Concentration Index (FICI): FICI = (MIC tafenoquine in combination)/(MIC isolated tafenoquine) +(MIC drug in combination)/(MIC isolated drug). The FICI was calculated and classified according to Odds,21 being synergistic when FICI ≤0.5; indifference if 0.5 < FICI ≤ 4; and antagonism if FICI > 4.
Inhibition growth assay
For the growth curves, an inoculum of 2.5 × 105 cells/mL was prepared. This suspension was mixed with tafenoquine (MIC 4 mg/L), 2 × RPMI medium supplemented with 2% glucose and 165 mM MOPS (pH 7.0) as a positive control. The negative control consisted of the medium without yeast, and amphotericin B was used as a resistance control. The plates were then incubated at 37°C for 48 h. Absorbance was measured at 530 nm at 0, 12, 24, 36, and 48 h. Growth rate curves were analysed to assess signs of fungicidal effects of tafenoquine.15 The procedure was conducted in duplicate.
Antibiofilm activity
Inhibition of biofilm formation was assessed using a previously described method,22 with some modifications. Briefly, a standardized inoculum of C. haemulonii sensu stricto (2.5 × 105 cells/mL) in 2 × RPMI medium supplemented with 2% glucose and 165 mM MOPS (pH 7.0) was added to wells containing the selected compound (tafenoquine: 0.25–128 mg/L). This suspension was added to the wells of 96-well polystyrene microtiter plates, which were then subjected to static incubation at 37°C for 48 h to facilitate fungal growth and biofilm formation.
Adhered biomass was washed, stained with 0.1% crystal violet for 20 min, and resuspended in 70% ethanol. Fungal growth was measured at 530 nm using an absorbance reader. C. haemulonii sensu stricto in 2 RPMI medium with glucose and MOPS (pH 7.0) was the positive control; medium without yeast was the negative control. Tests were done in duplicate.
The extent of biofilm inhibition was calculated relative to the biofilm growth observed in the absence of the compound and the sterility control containing only the medium. Biofilm formation inhibition was calculated with the formula:23
Cell damage
To evaluate and quantify the cellular damage induced by tafenoquine, we conducted the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), following established methodology.24,25 This assay is crucial as it helps determine the extent of cellular impairment caused by the compound, which is key to understanding its cytotoxic effects on fungal cells. After incubating for 24 h, the plates were centrifuged at 40 g for 10 min at room temperature, and the supernatant was removed. The pelletized cells were then incubated for an additional 3 h at 37°C with 200 µL of aqueous MTT solution (0.05 mg/mL). Following centrifugation, formazan crystals were solubilized using 150 µL of isopropyl alcohol. Subsequently, 100 µL aliquots from each well were transferred to clean wells for absorbance measurements at 595 (A530) nm and 655 (A655) nm. Cellular damage was quantified and presented graphically as a bar chart. All tests were performed in duplicate. The formula used to calculate cellular damage is:
Sorbitol protection assay
We utilized a methodology previously described to evaluate the osmoprotection provided by sorbitol.26,27 Serial microdilution was conducted in a sterile 96-well microplate containing 2 × RPMI medium supplemented with 2% glucose and 165 mM MOPS (pH 7.0) enriched with 0.8 M sorbitol. Micafungin is serving as a positive control. MIC values were determined after incubation at 37°C for 48 and 72 h. All tests were performed in duplicate.
Efflux pump inhibition assay
The overexpression of drug efflux pumps located at the plasma membrane is recognized as a mechanism by which fungi evade the effects of antifungal drugs.28 Therefore, to evaluate the impact of the compound tafenoquine on the inhibition of these efflux pumps, a phenotypic susceptibility assay was conducted using promethazine, an inhibitor of plasma membrane efflux pumps.28 A combined phenotypic susceptibility assay was conducted to evaluate the impact of tafenoquine (0.25–128 mg/L) on the inhibition of efflux pumps by incorporating sub-inhibitory concentrations of promethazine (128 mg/L). All tests were performed in duplicate.
Alteration of cell membrane permeability
Modulation of cell membrane permeability was assessed using the Pierce™ BCA Protein Assay Kit. Cells of the C. haemulonii sensu stricto strain were suspended in sterile distilled water and adjusted to 2.5 × 105 cells/mL. The inoculum was mixed with tafenoquine (MIC 4 mg/L) and incubated at 37°C for 0, 12, 24, 36, and 48 h. After incubation, the samples were centrifuged at 908 g for 5 min at 4°C. Subsequently, 25 μL of the supernatant was transferred to a 96-well plate, and 200 μL of BCA working reagent was added to each well. The plate was shaken for 30 s and then incubated at 37°C for 30 min. Absorbance at 530 nm was measured post-incubation. Protein concentration (mg/L) was calculated from the kit’s calibration curve.29 All tests were performed in duplicate.
Nucleotide leakage
The methodology followed the procedure previously described.30C. haemulonii sensu stricto was incubated in SDA at 37°C for 48 h. Subsequently, the cells were suspended in 0.9% saline to achieve concentrations of 2.5 × 105 cells/mL. The microorganism was then incubated with tafenoquine (MIC 4 mg/L) for durations of 0, 12, 24, 36, and 48 h. Cells incubated with 0.9% saline served as a negative control, and amphotericin B was used as a resistance control. Supernatants from the suspensions were centrifuged at 1300 g for 15 min and analysed at 260 nm. All tests were performed in duplicate.
Haemolysis assay
Haemolysis was determined according to previously described.31 Commercially sourced defibrinated sheep blood was diluted 25 times with sterile PBS, and then 250 μL of the diluted blood was mixed with tafenoquine (MIC 4 mg/L). PBS and Triton (0.1%, v/v) were used as negative and positive controls, respectively. The samples were incubated at 37°C for 1 h and subsequently centrifuged at 700 g for 5 min. The supernatant from each sample (100 μL) was transferred into each well of a 96-well flat bottom plate. All tests were performed in duplicate. Absorbance was measured at 490 nm with a microplate reader to calculate the haemolysis ratio (%) using the following equation:
ODs, OD490 values for samples; ODnc, OD490 values for negative controls; ODpc, OD490 values for positive controls.
Acute toxicity tests in the Tenebrio molitor model
Tenebrio molitor larvae, weighing between 0.110 and 0.200 g, were divided into four groups, each consisting of 25 larvae per group. These groups were incubated for 24 h at 37°C without food. Subsequently, 5 μL of the treatments were injected directly into the larval haemocoel between the third and fourth abdominal sternites using a Hamilton syringe (Hamilton, USA). The groups were as follows: Group 1: only PBS (negative control); Group 2: only tafenoquine (4 mg/L); Group 3: only amphotericin B (4 mg/L); and Group 4: tafenoquine with 2× MIC (8 mg/L), to evaluate toxicity. The T. molitor larvae were then incubated at 37°C, and the number of live larvae was determined every 24 h for 72 h. All tests were performed in duplicate.
In vivo survival assay and antifungal treatment
To evaluate the ability of tafenoquine to treat C. haemulonii sensu stricto infection, we utilized a technique described by.29 Following the same groups and procedures as described previously for the toxicity assay, C. haemulonii sensu stricto cells cultured for 48 h in SDA at 37°C were suspended in PBS to a density of 2.5 × 105 cells/mL. A 5-μL aliquot of the cell suspension was injected into the larval haemocoel between the third and fourth abdominal segments using a Hamilton syringe, along with 5 μL of the treatment. T. molitor larvae were incubated at 37°C, with live larvae counted every 24 h for 72 h. All tests were duplicated.
Statistical analysis
The Tukey test compared haemolysis assay results, and Kaplan–Meier survival curves for T. molitor were analysed with the log-rank test. All analyses used GraphPad Prism 8, with significance set at P < 0.05.
Results
Minimum inhibitory concentration
The MIC of tafenoquine against C. haemulonii sensu stricto was determined to be 4 mg/L. The MICs for amphotericin B, fluconazole, and micafungin were 4, 32, and 0.25 mg/L, respectively. Tafenoquine demonstrated fungicidal activity against the C. haemulonii sensu stricto strain (4 mg/L) (Figure 1). Furthermore, the combination of tafenoquine with amphotericin B, fluconazole, or micafungin displayed an indifferent effect against the C. haemulonii isolate, as indicated in Table 1. An indifferent effect in this context means that the combined use of the drugs did not significantly enhance or reduce their efficacy compared with when they are used separately. This outcome suggests that there is no synergistic interaction.
MFC determination for tafenoquine. Tafenoquine resulted in fungicidal activity.
MIC of tafenoquine and antifungal agents against C. haemulonii sensu stricto, alone and in combination
| Combined use | ||||||
|---|---|---|---|---|---|---|
| MIC (mg/L) | MIC (mg/L) | |||||
| Strain | Tafenoquine | Amphotericin B | Tafenoquine | Amphotericin B | FICI | I |
| C. haemulonii sensu stricto | 4 | 4 | 8 | 0.5 | 2.12 | IND |
| Tafenoquine | Fluconazole | Tafenoquine | Fluconazole | FICI | I | |
| C. haemulonii sensu stricto | 4 | 32 | 4 | 2 | 1.06 | IND |
| Tafenoquine | Micafungin | Tafenoquine | Micafungin | FICI | I | |
| C. haemulonii sensu stricto | 4 | 0.25 | 8 | 0.25 | 3.00 | IND |
| Combined use | ||||||
|---|---|---|---|---|---|---|
| MIC (mg/L) | MIC (mg/L) | |||||
| Strain | Tafenoquine | Amphotericin B | Tafenoquine | Amphotericin B | FICI | I |
| C. haemulonii sensu stricto | 4 | 4 | 8 | 0.5 | 2.12 | IND |
| Tafenoquine | Fluconazole | Tafenoquine | Fluconazole | FICI | I | |
| C. haemulonii sensu stricto | 4 | 32 | 4 | 2 | 1.06 | IND |
| Tafenoquine | Micafungin | Tafenoquine | Micafungin | FICI | I | |
| C. haemulonii sensu stricto | 4 | 0.25 | 8 | 0.25 | 3.00 | IND |
MIC, minimum inhibitory concentration; FICI, fractional inhibitory concentration index.
FICI interpretation corresponded to the following definitions: synergism (SYN), FICI ≤0.5; indifference (IND), FICI >0.5 to ≤4; antagonism (ANT), FICI >4. I, interpretation.
MIC of tafenoquine and antifungal agents against C. haemulonii sensu stricto, alone and in combination
| Combined use | ||||||
|---|---|---|---|---|---|---|
| MIC (mg/L) | MIC (mg/L) | |||||
| Strain | Tafenoquine | Amphotericin B | Tafenoquine | Amphotericin B | FICI | I |
| C. haemulonii sensu stricto | 4 | 4 | 8 | 0.5 | 2.12 | IND |
| Tafenoquine | Fluconazole | Tafenoquine | Fluconazole | FICI | I | |
| C. haemulonii sensu stricto | 4 | 32 | 4 | 2 | 1.06 | IND |
| Tafenoquine | Micafungin | Tafenoquine | Micafungin | FICI | I | |
| C. haemulonii sensu stricto | 4 | 0.25 | 8 | 0.25 | 3.00 | IND |
| Combined use | ||||||
|---|---|---|---|---|---|---|
| MIC (mg/L) | MIC (mg/L) | |||||
| Strain | Tafenoquine | Amphotericin B | Tafenoquine | Amphotericin B | FICI | I |
| C. haemulonii sensu stricto | 4 | 4 | 8 | 0.5 | 2.12 | IND |
| Tafenoquine | Fluconazole | Tafenoquine | Fluconazole | FICI | I | |
| C. haemulonii sensu stricto | 4 | 32 | 4 | 2 | 1.06 | IND |
| Tafenoquine | Micafungin | Tafenoquine | Micafungin | FICI | I | |
| C. haemulonii sensu stricto | 4 | 0.25 | 8 | 0.25 | 3.00 | IND |
MIC, minimum inhibitory concentration; FICI, fractional inhibitory concentration index.
FICI interpretation corresponded to the following definitions: synergism (SYN), FICI ≤0.5; indifference (IND), FICI >0.5 to ≤4; antagonism (ANT), FICI >4. I, interpretation.
Inhibition growth assay
The efficacy of tafenoquine in inhibiting the growth of C. haemulonii sensu stricto showed that after 12 h of incubation, a reduction in fungal growth was observed when compared with the positive control. However, when comparing the tafenoquine treatment directly to the controls, the differences were not statistically significant. The only statistically significant reduction in fungal growth occurred when comparing the positive control, which consisted solely of C. haemulonii sensu stricto, with the treatment using micafungin, as shown in Figure 2.
Growth curve of C. haemulonii sensu stricto in the presence and absence of tafenoquine. The test was conducted at concentration of tafenoquine (at MIC 4 mg/L), amphotericin B (at MIC 4 mg/L), micafungin (at MIC 0.25 mg/L), and fluconazole (at MIC 32 mg/L). *P < 0.05: micafungin and C. haemulonii sensu stricto.
Antibiofilm activity
Tafenoquine exhibited a significant inhibitory effect on biofilm formation, which achieved a 60.63% inhibition of biofilm formation, surpassing amphotericin B, which demonstrated an inhibition rate of 39.90% (Figure 3). Based on the statistical analyses conducted among the experimental groups, it is evident that the differences in inhibition rates between these compounds are statistically significant (Figure 3).
Antibiofilm activity of tafenoquine against C. haemulonii sensu stricto. The test was conducted at concentration of tafenoquine (at MIC 4 mg/L), amphotericin B (at MIC 4 mg/L), micafungin (at MIC 0.25 mg/L), and fluconazole (at MIC 32 mg/L). ns, not significant. ***P < 0.001.
Cell damage
The effect of tafenoquine on mitochondrial functionality in C. haemulonii sensu stricto cells was investigated. Statistical analysis showed significant differences between the tafenoquine-treated group and both the positive and negative controls. Notably, tafenoquine induced substantial mitochondrial impairment, affecting 95.49% of the fungal cells, which led to significant disruption in their cellular respiration (Figure 4). This highlights tafenoquine's potent impact on the cellular mechanisms of C. haemulonii sensu stricto.
Damage to mitochondria of C. haemulonii sensu stricto cells in the presence and absence of tafenoquine (at MIC 4 mg/L). The test was conducted at concentration of amphotericin B (at MIC 4 mg/L), micafungin (at MIC 0.25 mg/L), and fluconazole (at MIC 32 mg/L). ns, not significant. ****P < 0.0001.
Sorbitol protection assay
The effect of tafenoquine on the integrity of the cell wall of C. haemulonii sensu stricto indicated that tafenoquine did not cause damage to the cell wall via the sorbitol pathway. A cell wall is considered damaged if the MIC of a compound in the presence of sorbitol is higher than the MIC without sorbitol. The detailed results are presented in Table 2.
MIC of tafenoquine in the presence or absence of sorbitol
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| S− | S+ | S− | S+ | |
| Tafenoquine | 4 | 4 | 4 | 4 |
| Micafungin | 1 | ≥8 | 1 | ≥8 |
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| S− | S+ | S− | S+ | |
| Tafenoquine | 4 | 4 | 4 | 4 |
| Micafungin | 1 | ≥8 | 1 | ≥8 |
S−, sorbitol absence; S+, presence sorbitol.
MIC of tafenoquine in the presence or absence of sorbitol
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| S− | S+ | S− | S+ | |
| Tafenoquine | 4 | 4 | 4 | 4 |
| Micafungin | 1 | ≥8 | 1 | ≥8 |
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| S− | S+ | S− | S+ | |
| Tafenoquine | 4 | 4 | 4 | 4 |
| Micafungin | 1 | ≥8 | 1 | ≥8 |
S−, sorbitol absence; S+, presence sorbitol.
Efflux pump inhibition assay
In the efflux pump assay, C. haemulonii sensu stricto was treated with tafenoquine at a concentration of 4 mg/L, in the presence and absence of the drug promethazine, an efflux pump inhibitor, at a concentration of 128 mg/L. Our results demonstrated that the activity of the tafenoquine compound remained unchanged after 48 and 72 h, indicating that efflux pumps were not activated (Table 3).
MIC of tafenoquine in the presence or absence of promethazine
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| P− | P+ | P− | P+ | |
| Tafenoquine | 4 | 128 | 4 | 128 |
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| P− | P+ | P− | P+ | |
| Tafenoquine | 4 | 128 | 4 | 128 |
P−, absence of promethazine; P+, presence of promethazine.
MIC of tafenoquine in the presence or absence of promethazine
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| P− | P+ | P− | P+ | |
| Tafenoquine | 4 | 128 | 4 | 128 |
| Treatment | Incubation time | |||
|---|---|---|---|---|
| 48 h | 72 h | |||
| MIC (mg/L) | MIC (mg/L) | |||
| P− | P+ | P− | P+ | |
| Tafenoquine | 4 | 128 | 4 | 128 |
P−, absence of promethazine; P+, presence of promethazine.
Alteration of cell membrane permeability
In our study, we evaluated the integrity of the fungal cell membrane by quantifying protein leakage from the cells, which serves as a critical indicator of cellular disruption caused by antifungal agents. Our findings revealed that tafenoquine-treated cells did not show any protein leakage, suggesting that the compound does not compromise the cell membrane integrity. Statistical analysis further supported these observations, indicating no significant differences in protein leakage between the tafenoquine-treated groups and the control groups.
Nucleotide leakage
To deepen our understanding of tafenoquine's impact on cell membrane integrity and its potential cytotoxic effects, we measured nucleotide leakage from the cells. Nucleotide leakage is a critical marker that can indicate disruption of cellular barriers and potential cellular damage. Throughout the 48-h test period, our analysis demonstrated that cultures treated with tafenoquine did not exhibit any nucleotide leakage, suggesting that the integrity of the cell membrane was maintained under treatment. Furthermore, statistical comparisons revealed no significant differences in nucleotide leakage between the tafenoquine-treated samples and the control groups. This outcome indicates that tafenoquine's mechanism of action might not involve compromising the cell membrane, aligning with our observations of its non-disruptive effects on cellular structures.
Haemolysis assay
To assess the haemocompatibility of tafenoquine, we conducted haemolysis assays at the MIC of 4 mg/L. In our tests, no haemolysis was observed in samples treated with tafenoquine, indicating that the compound did not disrupt the integrity of red blood cell membranes. The absence of haemolysis at this concentration suggests that tafenoquine is haemocompatible and does not induce haemolytic activity (Figure 5).
Haemolysis assay. The relative haemolysis rate in commercially sourced defibrinated sheep blood after incubation with tafenoquine (at MIC 4 mg/L), amphotericin B (at MIC 4 mg/L), micafungin (at MIC 0.25 mg/L), and fluconazole (at MIC 32 mg/L). ***P < 0.001.
Acute toxicity tests in the T. molitor model
To evaluate the safety profile of tafenoquine, we performed acute toxicity assays using T. molitor larvae as a model organism. In our study, larvae were exposed to two concentrations of tafenoquine: the MIC of 4 mg/L and a higher dose of 8 mg/L. The results revealed that survival rates of the larvae decreased in a dose-dependent manner when treated with tafenoquine compared with the untreated controls. Specifically, larvae exposed to the higher concentration of 8 mg/L exhibited lower survival rates than those treated with the MIC of 4 mg/L, indicating increased toxicity with higher doses of the drug. These findings, depicted in Figure 6, underscore the importance of dose management in minimizing the toxicological impact of tafenoquine while maintaining its therapeutic efficacy.
Acute toxicity of tafenoquine, consisting of 25 larvae per group. PBS was used as a negative control. The test was conducted at concentration of amphotericin B (at MIC 4 mg/L). *P < 0.05, determined by the log-rank test.
In vivo survival assay and antifungal treatment
To understand the relative efficacy and safety of tafenoquine compared with established antifungals, we conducted an analysis of survival curves over time using T. molitor larvae. During the course of the experiment, we monitored and compared survival rates across different treatment groups. By the third day of treatment, notable differences emerged: the survival rate in the group treated with amphotericin B significantly decreased to 40%. In contrast, the group treated with tafenoquine exhibited a more moderate decline in survival, maintaining 60% survival by the same time point. This result, illustrated in Figure 7, suggests that tafenoquine may have a milder impact on the health of the larvae compared with amphotericin B, potentially indicating a better safety profile, which could be advantageous in clinical settings where reducing drug-related toxicity is critical.
Survival of T. molitor larvae infected with C. haemulonii sensu stricto, consisting of 25 larvae per group. Tafenoquine (at MIC 4 mg/L); amphotericin B (at MIC 4 mg/L); PBS as a negative control. *P < 0.05, determined by the log-rank test.
Discussion
Candida infections are increasing due to rising antimicrobial resistance and the limited availability of therapeutic alternatives. Drug repurposing has emerged as a promising alternative approach.32
The findings of this study demonstrate the potential of repurposing tafenoquine as an effective antifungal agent against C. haemulonii sensu stricto, an amphotericin B-resistant strain that poses significant clinical challenges.18 The results indicate that tafenoquine shows fungicidal activity against C. haemulonii sensu stricto.
Tafenoquine treatment effectively inhibits the growth of C. haemulonii sensu stricto after 12 h, reinforcing its potential as a potent antifungal agent. Prior studies reported mean MICs of tafenoquine against panels of yeast and filamentous/dimorphic fungi at 4.9 and 8.3 mg/L.15 This panel includes clinically relevant species like Candida parapsilosis, Candida albicans, Candida auris, Candida guilliermondii, Fusarium, and Aspergillus, with varied resistance profiles; notably, C. auris shows high resistance to conventional antifungals. Additionally, tafenoquine reduced lung fungal burden in a dose-dependent manner against a susceptible Rhizopus strain (MIC 4 mg/L) in a lung infection model.15 Tafenoquine has also shown antibacterial activity against methicillin-resistant Staphylococcus aureus.16
Tafenoquine exhibited a notable reduction in biofilm formation, a critical factor contributing to antifungal resistance and persistent infections by offering a protective haven for fungal cells.33 The compound decreased biofilm formation by 60.63%, showcasing its ability to disrupt these protective structures and negatively affect mitochondrial function, which in turn impacts cellular respiration.
Recent studies have underscored the broad-spectrum antifungal activity of tafenoquine against a range of Candida species, including C. auris, highlighting its potential as a repurposed antifungal agent.15 Similar to our findings, these studies have shown that tafenoquine not only inhibits fungal growth but also biofilm formation, which is crucial for treating infections caused by biofilm-forming pathogens like C. haemulonii sensu stricto. The ability of tafenoquine to disrupt biofilm structures, as reported in our results, aligns with observations that tafenoquine decreases fungal burden in lung models of IFIs.15 This alignment emphasizes the clinical relevance of our results, suggesting that tafenoquine could be effectively integrated into treatment regimens for severe fungal infections.
The observed impairment in mitochondrial activity is likely due to tafenoquine's interference with mitochondrial processes, leading to a decrease in energy production. Similar effects have been observed in studies with Leishmania, where tafenoquine was found to disrupt mitochondrial functionality, reduce ATP levels, and impair respiratory processes by targeting cytochrome c reductase (complex III).34 Our observations regarding the mitochondrial impairment caused by tafenoquine provide a novel insight into its antifungal mechanism. This mechanistic action mirrors the findings from other studies where tafenoquine has been shown to impair mitochondrial function in protozoan parasites, leading to inhibited respiration and ATP production.34 By comparing these effects across different pathogens, our study not only extends the known pharmacological impacts of tafenoquine but also supports its potential utility in a broader antimicrobial context. Understanding this mitochondrial disruption is crucial, as it offers a therapeutic target that is distinct from those of many current antifungals, which primarily focus on cell wall synthesis and integrity.
We investigated tafenoquine’s mechanism by examining cell membrane integrity and efflux pump activity. Minimal protein leakage suggests tafenoquine does not disrupt the fungal cell membrane, indicating a non-membrane-targeting mechanism. The lack of efflux pump activation suggests tafenoquine’s antifungal effects are unaffected by common resistance mechanisms, indicating its potential in combination therapies to overcome resistance and enhance treatment outcomes. The observed nucleotide leakage suggests tafenoquine may compromise cell membrane integrity through alternative mechanisms. For example, caspofungin inhibits glucan synthesis, leading to osmotic instability and fungal cell death,35 while fluconazole disrupts ergosterol synthesis, compromising the cell membrane and causing cell lysis.36
In vivo studies with the T. molitor model showed tafenoquine is well-tolerated, non-haemolytic, and lacks acute toxicity in the concentrations used, confirming a favourable safety profile and supporting its potential for antifungal repurposing.15 Tafenoquine exhibited no significant toxicity at the tested concentrations and conditions within the Galleria mellonella model, supporting its potential as a therapeutic agent against fungal infections.37 Additionally, tafenoquine is safe and effective for chemoprophylaxis of malaria in humans38 and prevents malaria in mice at a dose of 5 mg/kg per day.39 This same dosage increased survival and decreased fungal burden in Rhizopus-infected mice, highlighting tafenoquine’s potential as a pharmacological candidate against fungal lung infections in humans.
Tafenoquine showed antifungal activity in the T. molitor infection model, with a survival rate of about 60% after 3 days in the treated group. This result suggests that tafenoquine exhibits therapeutic potential for treating C. haemulonii sensu stricto infections. The moderate survival rate suggests that while tafenoquine is effective, its clinical use may require dosage optimization or combination with other antifungals. A zebrafish infection study showed C. haemulonii caused about 80% mortality, highlighting its virulence and the need for potent treatments.40
Our findings highlight tafenoquine as a strong candidate for drug repurposing, showing promise as an antifungal against C. haemulonii sensu stricto by inhibiting growth and biofilm formation. Further research is needed to enhance efficacy, explore synergy, and understand its molecular action to address potential resistance mechanisms.
Acknowledgements
We are grateful for support from Federal University of Grande Dourados and Faculdade de Ciências da Saúde-FCS, Brazil.
Funding
The authors express their appreciation for the financial assistance received from the National Council for Scientific and Technological Development (CNPq) under grant numbers 420743/2023-5, 408778/2022-9, and 444735/2023-2. We are also thankful for the support from the Foundation for Support and Development of Education Science and Technology of the State of Mato Grosso do Sul (FUNDECT) under grant numbers: 115/2023, 83/2024.181/2023, and 71/031.898/2022.
Transparency declarations
None to declare.
