Identification of Promising Antifungal Drugs against Scedosporium and Lomentospora Species after Screening of Pathogen Box Library

Fungal infections have been increasing during the last decades. Scedosporium and Lomentospora species are filamentous fungi most associated to those infections, especially in immunocompromised patients. Considering the limited options of treatment and the emergence of resistant isolates, an increasing concern motivates the development of new therapeutic alternatives. In this context, the present study screened the Pathogen Box library to identify compounds with antifungal activity against Scedosporium and Lomentospora. Using antifungal susceptibility tests, biofilm analysis, scanning electron microscopy (SEM), and synergism assay, auranofin and iodoquinol were found to present promising repurposing applications. Both compounds were active against different Scedosporium and Lomentospora, including planktonic cells and biofilm. SEM revealed morphological alterations and synergism analysis showed that both drugs present positive interactions with voriconazole, fluconazole, and caspofungin. These data suggest that auranofin and iodoquinol are promising compounds to be studied as repurposing approaches against scedosporiosis and lomentosporiosis.


Introduction
Fungal infections have been emerging during the last decades as a consequence of increasing numbers of individuals suffering from health problems, such as diabetes, chemotherapy for cancer treatment, HIV/AIDS, and other immunosuppressive conditions [1,2]. In this context, Scedosporium and Lomentospora species constitute a relevant group of filamentous fungi that cause a wide range of clinical manifestations, being considered emergent pathogens since its incidence increased in the last decades in Europe, America, Asia, and Oceania [3,4]. Scedosporiosis and lomentosporiosis are usually associated with organ transplant recipients, near-drowning people, and HIV/AIDS patients, in which invasive infections can be observed [5]. In addition, they are the second most frequent cause of pulmonary fungal infections in cystic fibrosis patients [6]. In immunocompetent patients, Scedosporium and Lomentospora species cause superficial and cutaneous infections, such as mycetoma, which can lead to the amputation of the affected member in the absence of suitable treatment [3].
The treatment of fungal infections, including scedosporiosis and lomentosporiosis, is limited due to the few antifungal drugs available in clinical settings. Currently, only three classes of antifungals are commonly used to treat mycoses, such as polyenes that directly were maintained in modified Sabouraud medium (0.5% yeast extract, 1% peptone, and 2% glucose monohydrate). To obtain conidia, cells were grown on plates containing modified Sabouraud agar medium for seven days at room temperature. After that, the surface of the medium was washed with sterile phosphate-buffered saline (PBS, pH 7.2), and the conidia were removed with the aid of a sterile spatula. The cell suspension was filtered and later centrifuged to be used in the experiments.

Compounds
The Medicines for Malaria Venture organization provided the Pathogen Box library, which is composed of 400 compounds at 10 mm in dimethyl sulfoxide (DMSO). A stock solution of each compound was kept at 1 mm in DMSO and stored at −20 • C. Additional experiments were conducted using auranofin and iodoquinol powder (Sigma Chemical Co., St. Louis, MO, USA) dissolved in DMSO and stored at −20 • C, as well as voriconazole, fluconazole, and caspofungin (Sigma Chemical Co., St. Louis, MO, USA).

Screening of the Pathogen Box Library
The reference isolate S. aurantiacum CBS 136046 was used to screen the Pathogen Box library due to its relevance as a virulent and resistant species from the Scedosporium and Lomentospora groups.
Screening was performed in 96-well microtiter plates containing a final concentration of 5 µm of each compound diluted in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 2% glucose and buffered with 3-(N-morpholino) propanesulphonic acid (MOPS) (0.165 mol/L, pH 7.2, from here on referred to as 'supplemented RPMI'). Voriconazole at 5 µm and RPMI supplemented with DMSO 1% were used as controls. Conidia (2 × 10 5 /mL) were added and incubated for 72 h at 37 • C in a 5% CO 2 atmosphere. Fungal growth was analyzed by visual inspection and quantified by optical density readings using a spectrophotometer (Bio-Rad, Hercules, CA, USA) at 600 nm. An inhibition of at least 80% was defined as a cut-off to select the promising drugs with antifungal activity against Scedosporium and Lomentospora species.

Antifungal Susceptibility Testing
The susceptibility of Scedosporium and Lomentospora species to auranofin and iodoquinol was determined by the broth microdilution method, according to EUCAST protocols, with modifications [43]. Voriconazole was also included in experiments as a reference antifungal because it is the drug of choice for the treatment of scedosporiosis. Briefly, compounds were serially diluted (10-0.078 µm) in supplemented RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 2% glucose and buffered with 3-(N-morpholino) propanesulphonic acid (MOPS) (0.165 mol/L, pH 7.2) in 96-well microplates. A standardized suspension of conidia (2 × 10 5 /mL) was added in microplates and incubated for 72 h at 37 • C, in a 5% CO 2 atmosphere. Fungal growth was analyzed by spectrophotometry readings (Bio-Rad, Hercules, CA, USA) at 600 nm and cell viability was assessed using the XTT-reduction assay [44]. The minimum inhibitory concentration (MIC) of each compound was defined as the lowest concentration that inhibits 80% of fungal growth.

Biofilm Formation and the Preformed Biofilm Assay
Biofilm formation was analyzed according to [36]. Briefly, 200 µL from a standardized suspension of Scedosporium and Lomentospora conidia (1 × 10 7 /mL) was added to each well of a polystyrene microplate and incubated for 1.5 h at 37 • C for the adhesion step. After that, the supernatant containing non-adherent cells was removed and RPMI 1640 medium supplemented with MOPS, 2% glucose, and 20% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) was added in the absence (positive control) or presence of selected compounds (8-0.25 × MIC). Adherent cells were then incubated for 24 h at 37 • C. For the preformed biofilm assay, cells were cultured to form biofilm as described above in the absence of the compounds. After 24 h of biofilm formation, the supernatant was removed, and supplemented RPMI was added in the absence (positive control) or presence of the selected compounds (8-0.25 × MIC). An additional incubation of 24 h at 37 • C was performed to evaluate the anti-biofilm activity. Both biofilm formation and preformed biofilms were evaluated using three parameters as previously described [44][45][46]. Crystal violet, safranin, and XTT assays were used to analyze the overall biomass, extracellular matrix, and metabolic activity, respectively. adhesion to aluminum stubs with carbon tape; and viii.
coating with gold.
Images were obtained with FEI Quanta 250 scanning electron microscope (FEI Company, Hillsboro, OR, USA) and processed using Photoshop software (Adobe, San José, CA, USA).
Measurement of hyphae thickness was performed using the software ImageJ from National Institutes of Health (NIH).

Antifungal Drug Synergy Assay
Synergistic interactions were evaluated by the checkerboard method according to EU-CAST guidelines [47]. S. aurantiacum conidia (1 × 10 5 /mL) were grown in 96-well plates containing supplemented RPMI in the presence of selected compounds (0.156-10 µm) combined with fluconazole (5-320 µm), voriconazole (0.47-30 µm), or caspofungin (0.625-40 µm). After incubation for 72 h at 37 • C, MIC was evaluated at 600 nm and cell viability was assessed by the XTT-reduction assay at 490 nm using a spectrophotometer (Bio-Rad, Hercules, CA, USA). An inhibition of at least 80% was defined as a cut-off for minimum inhibitory concentration (MIC). Minimum effective concentration (MEC) was used to assess caspofungin activity and its interaction with auranofin and iodoquinol, since MEC values are considered more suitable for echinocandins analysis [48]. Interactions were determined by two different methods, the fractional inhibitory concentration index (FICI) and the Bliss independence model.
Bliss independence model was performed according to Meletiadis and colleagues and Zhao and colleagues [50,51]. The following formula was used to assess the drug interaction: in which E exp is the expected efficacy of drug combination, E a is the efficacy of drug A (auranofin or iodoquinol), and E b is the efficacy of drug B (fluconazole, voriconazole or caspofungin). The results were classified as: synergistic effect, E obs > E exp ; indifference, E obs = E exp ; antagonistic effect, E obs < E exp .

Statistical Analyses
All experiments were performed in triplicate, in three independent experimental sets. Statistical analyses were performed using GraphPad Prism v5.00 for Windows (GraphPad Software, San Diego, CA, USA). The nonparametric Kruskal-Wallis one-way analysis of variance was used to compare the differences among the groups, and individual comparisons of the groups were performed using a Bonferroni post-test. The 90% or 95% confidence interval was determined in all experiments.

Screening of Pathogen Box Library
A total of 400 compounds from the Pathogen Box library were tested against S. aurantiacum as reference strain due to its relevance as a highly virulent and resistant species of Scedosporium group [4,52]. The screening revealed six compounds with antifungal activity at 5 µm, which induced at least 80% of S. aurantiacum inhibition ( Figure 1). Voriconazole was used as a control, inducing inhibition of 89.08%.
Bliss independence model was performed according to Meletiadis and colleagues and Zhao and colleagues [50,51]. The following formula was used to assess the drug interaction: Eexp = Ea + Eb − Ea × Eb, in which Eexp is the expected efficacy of drug combination, Ea is the efficacy of drug A (auranofin or iodoquinol), and Eb is the efficacy of drug B (fluconazole, voriconazole or caspofungin). The results were classified as: synergistic effect, Eobs > Eexp; indifference, Eobs = Eexp; antagonistic effect, Eobs < Eexp.

Statistical Analyses
All experiments were performed in triplicate, in three independent experimental sets. Statistical analyses were performed using GraphPad Prism v5.00 for Windows (GraphPad Software, San Diego, CA, USA). The nonparametric Kruskal-Wallis one-way analysis of variance was used to compare the differences among the groups, and individual comparisons of the groups were performed using a Bonferroni post-test. The 90% or 95% confidence interval was determined in all experiments.

Screening of Pathogen Box Library
A total of 400 compounds from the Pathogen Box library were tested against S. aurantiacum as reference strain due to its relevance as a highly virulent and resistant species of Scedosporium group [4,52]. The screening revealed six compounds with antifungal activity at 5 μm, which induced at least 80% of S. aurantiacum inhibition ( Figure 1). Voriconazole was used as a control, inducing inhibition of 89.08%. The identification of these six compounds is presented in Table 1. Two of them are known antifungal drugs, difenoconazole, and posaconazole. Another two are non-commercial molecules, 5-Chloro-6-[(2,5-dimethoxyanilino)methyl]quinazoline-2,4-diamine (which has already been described with an anti-cryptosporidiosis activity) and N-[3,4-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (which has already been described with an anti-tuberculosis activity). Finally, the last two compounds, auranofin and iodoquinol, are known drugs that are already used in clinical settings for the treatment of rheumatoid arthritis and amoebiasis, respectively. The identification of these six compounds is presented in Table 1. Two of them are known antifungal drugs, difenoconazole, and posaconazole. Another two are noncommercial molecules, 5-Chloro-6-[(2,5-dimethoxyanilino)methyl]quinazoline-2,4-diamine (which has already been described with an anti-cryptosporidiosis activity) and N-[3,4-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (which has already been described with an anti-tuberculosis activity). Finally, the last two compounds, auranofin and iodoquinol, are known drugs that are already used in clinical settings for the treatment of rheumatoid arthritis and amoebiasis, respectively. Considering that auranofin and iodoquinol are known drugs already used for other pathologies, we decided to select them for subsequent experiments due to their promising application as repurposing drugs to treat scedosporiosis and lomentosporiosis. The chemical structures of auranofin and iodoquinol are depicted in Figure 2.

Minimum Inhibitory Concentration of Auranofin and Iodoquinol against Different Scedosporium and Lomentospora Species
Since the screening of the Pathogen Box library was performed using only one concentration of each compound (5 μm), we evaluated the MIC of auranofin and iodoquinol as well as that of voriconazole used as a reference drug. The assay was performed not only against S. aurantiacum, but also against other clinically relevant species, such as S. boydii, S. apiospermum, S. dehoogii, and L. prolificans.
Auranofin displayed MIC of 5 μm for all five species tested and fungal viability was also inhibited at 5 μm, except for S. apiospermum whose viability was inhibited at 10 μm (Table 2). Iodoquinol presented MIC values of 5 μm for S. aurantiacum, 0.625 μm for S. boydii and L. prolificans, and 1.25 μm for S. apiospermum and S. dehoogii. Regarding the inhibition of fungal viability, iodoquinol was active at 5 μm for S. aurantiacum and S. apiospermum, 0.625 μm for S. boydii and L. prolificans, and 1.25 μm for S. dehoogii (Table 2).
Comparing these results with voriconazole, auranofin presented 12-fold lower MIC values for L. prolificans, whereas iodoquinol displayed lower values for most species (except for S. aurantiacum) ( Table 2).

Minimum Inhibitory Concentration of Auranofin and Iodoquinol against Different Scedosporium and Lomentospora Species
Since the screening of the Pathogen Box library was performed using only one concentration of each compound (5 µm), we evaluated the MIC of auranofin and iodoquinol as well as that of voriconazole used as a reference drug. The assay was performed not only against S. aurantiacum, but also against other clinically relevant species, such as S. boydii, S. apiospermum, S. dehoogii, and L. prolificans.
Auranofin displayed MIC of 5 µm for all five species tested and fungal viability was also inhibited at 5 µm, except for S. apiospermum whose viability was inhibited at 10 µm (Table 2). Iodoquinol presented MIC values of 5 µm for S. aurantiacum, 0.625 µm for S. boydii and L. prolificans, and 1.25 µm for S. apiospermum and S. dehoogii. Regarding the inhibition of fungal viability, iodoquinol was active at 5 µm for S. aurantiacum and S. apiospermum, 0.625 µm for S. boydii and L. prolificans, and 1.25 µm for S. dehoogii (Table 2). Comparing these results with voriconazole, auranofin presented 12-fold lower MIC values for L. prolificans, whereas iodoquinol displayed lower values for most species (except for S. aurantiacum) ( Table 2).

Effect of Auranofin and Iodoquinol on Fungal Biofilms
Auranofin and iodoquinol were also checked against biofilm formation and preformed biofilms of Scedosporium and Lomentospora species. Regarding preformed biofilms, auranofin decreased the fungal biomass about 50% of at 1 × MIC for S. aurantiacum and 70% for S. dehoogii and L. prolificans. For S. boydii and S. apiospermum, a maximum inhibition of 40% was observed at 8 × MIC ( Figure 3A, Table S1). Extracellular matrix was reduced to 50% at 1 × MIC for all fungi ( Figure 3B, Table S1), and biofilm viability decreased to less than 50% at 1 × MIC, reaching only 10% of viability at 8x MIC ( Figure 3C, Table S1). inhibition of 40% was observed at 8 × MIC ( Figure 3A, Table S1). Extracellular matrix was reduced to 50% at 1 × MIC for all fungi ( Figure 3B, Table S1), and biofilm viability decreased to less than 50% at 1 × MIC, reaching only 10% of viability at 8x MIC ( Figure 3C, Table S1). Iodoquinol did not reduce the biomass of preformed biofilms of S. boydii, S. apiospermum and S. dehoogii, but a reduction of more than 50% was observed for S. aurantiacum and L. prolificans at 4 × MIC ( Figure 3D, Table S1). Extracellular matrix was decreased about 50% at 4 × MIC, except for S. dehoogii, whose matrix was reduced only 35% at 4 × MIC ( Figure 3E, Table S1). Biofilm viability was 50% decreased at 2 × MIC for S. boydii and S. dehoogii, and 4 × MIC for S. apiospermum. For S. aurantiacum and L. prolificans, biofilm viability was found to be only 30% at 2 × MIC and 20% at 8 × MIC ( Figure 3F, Table S1). All data suggest that both compounds, auranofin and iodoquinol, were able to affect mature biofilms, especially their viability. Regarding the biofilm formation, a stronger overall effect was observed for both compounds. Auranofin caused 90% inhibition of biomass and extracellular matrix formation, as well as viability for all five species, except for L. prolificans whose viability was maintained between 30-50% at 1-8 × MIC ( Figure 4A-C, Table S2). Considering iodoquinol, 1 × MIC caused 90% inhibition of biomass and extracellular matrix formation, and Iodoquinol did not reduce the biomass of preformed biofilms of S. boydii, S. apiospermum and S. dehoogii, but a reduction of more than 50% was observed for S. aurantiacum and L. prolificans at 4 × MIC ( Figure 3D, Table S1). Extracellular matrix was decreased about 50% at 4 × MIC, except for S. dehoogii, whose matrix was reduced only 35% at 4 × MIC ( Figure 3E, Table S1). Biofilm viability was 50% decreased at 2 × MIC for S. boydii and S. dehoogii, and 4 × MIC for S. apiospermum. For S. aurantiacum and L. prolificans, biofilm viability was found to be only 30% at 2 × MIC and 20% at 8 × MIC ( Figure 3F, Table S1). All data suggest that both compounds, auranofin and iodoquinol, were able to affect mature biofilms, especially their viability.
Regarding the biofilm formation, a stronger overall effect was observed for both compounds. Auranofin caused 90% inhibition of biomass and extracellular matrix formation, as well as viability for all five species, except for L. prolificans whose viability was maintained between 30-50% at 1-8 × MIC ( Figure 4A-C, Table S2). Considering iodoquinol, 1 × MIC caused 90% inhibition of biomass and extracellular matrix formation, and viability for S. apiospermum, S. dehoogii, and L. prolificans (except the viability of L. prolificans, which was 75% inhibited at 8 × MIC) ( Figure 4D-F, Table S2). For S. aurantiacum and S. boydii, similar effects were only observed at 2 × MIC ( Figure 4D-F, Table S2).

Alterations Caused by Auranofin and Iodoquinol on S. aurantiacum Morphology
Considering that auranofin and iodoquinol presented interesting antifungal activity against planktonic cells and biofilms of different Scedosporium and Lomentospora species, we evaluated the alterations caused by both drugs on fungal morphology through scanning electron microscopy (SEM). S. aurantiacum was used as a reference species.
SEM analysis revealed that untreated S. aurantiacum had septated hyphae ( Figure  5A,B) with sympodial conidia showing an ellipsoidal shape ( Figure 6A,B). The treatment of S. aurantiacum with 2.5 μm auranofin (0.5 × MIC) induced alterations in the fungal cell wall integrity ( Figure 5C,D), while the treatment with iodoquinol also disrupts the cell wall ( Figure 6C,D) and increases the thickness of conidia ( Figure 6E).  E) and viability (C,F) were measured using violet crystal, safranin and XTT-reduction assay, respectively. * p < 0.01, compared to 0 (absence of drug) for each species.
All these data suggest that both auranofin and iodoquinol displayed a more drastic effect on biofilm formation compared to mature ones.

Alterations Caused by Auranofin and Iodoquinol on S. aurantiacum Morphology
Considering that auranofin and iodoquinol presented interesting antifungal activity against planktonic cells and biofilms of different Scedosporium and Lomentospora species, we evaluated the alterations caused by both drugs on fungal morphology through scanning electron microscopy (SEM). S. aurantiacum was used as a reference species. SEM analysis revealed that untreated S. aurantiacum had septated hyphae ( Figure 5A,B) with sympodial conidia showing an ellipsoidal shape ( Figure 6A,B). The treatment of S. aurantiacum with 2.5 µm auranofin (0.5 × MIC) induced alterations in the fungal cell wall integrity ( Figure 5C,D), while the treatment with iodoquinol also disrupts the cell wall ( Figure 6C,D) and increases the thickness of conidia ( Figure 6E).

Drug Interaction among Auranofin and Iodoquinol with Fluconazole, Voriconazole and Caspofungin
To evaluate the interaction properties of auranofin and iodoquinol with some current antifungal drugs used in clinical settings, a synergy analysis was performed. Once again, S. aurantiacum was used as a representative species. Drug interaction was analyzed between auranofin or iodoquinol with fluconazole, voriconazole, or caspofungin.
The MIC observed for fluconazole was reduced two-fold and four-fold after coincubation with auranofin and iodoquinol, respectively, while MIC for voriconazole was reduced two-fold when combined with iodoquinol. The decrease in the caspofungin MEC was more prominent after combination with iodoquinol (16-fold), but it was also observed with auranofin (4-fold) ( Table 3). Following the FIC index criteria, a synergism effect was observed between iodoquinol and caspofungin (FICI = 0.18) ( Table 3).

Drug Interaction among Auranofin and Iodoquinol with Fluconazole, Voriconazole and Caspofungin
To evaluate the interaction properties of auranofin and iodoquinol with some current antifungal drugs used in clinical settings, a synergy analysis was performed. Once again, S. aurantiacum was used as a representative species. Drug interaction was analyzed between auranofin or iodoquinol with fluconazole, voriconazole, or caspofungin.
The MIC observed for fluconazole was reduced two-fold and four-fold after co-incubation with auranofin and iodoquinol, respectively, while MIC for voriconazole was reduced two-fold when combined with iodoquinol. The decrease in the caspofungin MEC was more prominent after combination with iodoquinol (16-fold), but it was also In addition, a greater reduction in fungal viability after a combination of auranofin and iodoquinol with antifungal drugs was detected, compared with the activity observed using the same concentration of antifungal alone. Auranofin increased the caspofungin activity at 1.25 and 2.5 µm (0.25 and 0.5 × MIC, respectively), whereas it increased the fluconazole and voriconazole activities at 2.5 µm (0.5 × MIC) ( Figure 7A). Iodoquinol also potentiates the effectiveness of caspofungin (at all tested concentrations), fluconazole, and voriconazole (at 2.5 µm) ( Figure 7B). fluconazole and voriconazole activities at 2.5 μm (0.5 × MIC) ( Figure 7A). Iodoquinol also potentiates the effectiveness of caspofungin (at all tested concentrations), fluconazole, and voriconazole (at 2.5 μm) ( Figure 7B). Regarding the analysis using the Bliss independence method, auranofin presented a synergistic effect when combined with caspofungin and, to a low extent, with fluconazole and voriconazole (Table 4). Iodoquinol displayed synergistic interaction only with caspofungin (Table 4). Table 3. Antifungal activity of auranofin, iodoquinol, fluconazole, voriconazole and caspofungin-Alone and in combinations according to Fractional Inhibitory Concentration Index-against S. aurantiacum CBS 136046. MIC values were used to analyze the interaction between auranofin and iodoquinol with azoles (fluconazole and voriconazole), whereas MEC values were used to assess the interaction between auranofin and iodoquinol with caspofungin.   Regarding the analysis using the Bliss independence method, auranofin presented a synergistic effect when combined with caspofungin and, to a low extent, with fluconazole and voriconazole (Table 4). Iodoquinol displayed synergistic interaction only with caspofungin (Table 4).

Discussion
Scedosporiosis is a widespread infection that affects healthy and immunocompromised patients, causing superficial and invasive infections, respectively [3]. Scedosporiosis is associated with a variety of base conditions, such as cancer, hematological malignancies, organ transplantation, and AIDS [3,53,54]. The mortality rate reaches 75% in HIV patients [55]. In addition, Scedosporium and Lomentospora pathogens are known as one of the most frequent fungi colonizing lungs of cystic fibrosis patients [6,56].
Scedosporium and Lomentospora species are resistant to the most frequently administered antifungal agents, such as amphotericin B and different azoles and echinocandins [10]. In this context, S. aurantiacum has revealed itself as a highly virulent and resistant species [57], which is one reason why it was chosen as a representative species for the screening of the Pathogen Box library. Considering the concerns raised by Scedosporium pathogens in clinical settings, which include the hard-to-treat aspect of scedosporiosis and the high mortality levels especially in immunocompromised patients, the study of new treatment alternatives is an urgent need.
In the present study, we screened the Pathogen Box library against S. aurantiacum as a representative species of Scedosporium and Lomentospora group. This library was developed by the Medicines for Malaria Venture organization, which contains 400 compounds including new molecules and repurposing potential drugs (https://www.mmv.org/mmv-open/ pathogen-box) (accessed on 3 August 2021). Pathogen Box was developed to comprise promising compounds that might be active against pathogens associated with neglected diseases [37], which makes it a useful tool to search for new treatment alternatives for fungal infections.
Our screening revealed six compounds that displayed at least 80% inhibition against S. aurantiacum. Two known antifungal drugs (difenoconazole and posaconazole), two new molecules (MMV675968 and MMV687807), and two drugs already used for other pathologies (auranofin and iodoquinol). Pathogen Box has also been screened for other pathogenic fungi, such as Candida, Cryptococcus, and Sporothrix species, as well as chromoblastomycosis agents [38][39][40][41][42]. These studies observed that MMV675968 and MMV687807 were also active against Candida albicans and Sporothrix species [38,42], auranofin inhibited C. albicans and chromoblastomycosis agents [38,41], and iodoquinol displayed antifungal activity against Sporothrix species, chromoblastomycosis agents, and Candida auris [40][41][42]. Considering the potential of auranofin and iodoquinol to be used as a repurposing approach to treat fungal infections, we decided to select these two molecules to continue our analyses.
As mentioned above, auranofin and iodoquinol have already been evaluated against other pathogenic fungi. Whereas the antifungal activity of iodoquinol has only been demonstrated recently by studies using the Pathogen Box [40][41][42]58], the antifungal effect of auranofin has already been described in the literature for a variety of fungal pathogens, such as Candida species, C. neoformans, Blastomyces dermatitidis, Aspergillus fumigatus, and Rhizopus oryzae [59,60]. It has also been shown that auranofin is a promising repurposing drug to treat Scedosporium and Lomentospora infections, presenting minimal inhibitory concentrations ranging from 2 to >16 µg/mL [61].
Auranofin and iodoquinol presented antifungal activity not only against S. aurantiacum, but also against other species, suggesting that both drugs displayed a conserved effect in Scedosporium and Lomentospora group. In addition, auranofin displayed an anti-biofilm effect against different species of Scedosporium and Lomentospora groups, whereas iodoquinol was less active especially against preformed biofilms. In C. albicans, auranofin also displayed anti-biofilm activity [62,63], suggesting that it might be effective not only against planktonic growth, but also with adherent cells. Regarding iodoquinol, it also presented low activity against the preformed biofilm of C. auris [40], suggesting that more studies are needed to clarify the effect of this molecule on fungal biofilms. SEM analysis revealed fungal surface alterations when S. aurantiacum was exposed to auranofin. Its mechanism of action is described to involve the inhibition of small redox proteins called thioredoxin reductases (TrxR), which are responsible for maintaining a reduced cellular environment [64]. TrxR is present in a variety of cell models, such as mammalian cells, protozoans, bacteria, plants, and fungi, and its inhibition by auranofin has already been demonstrated in different cell models, including Entoameba histolytica, Staphylococcus aureus, and fungi [63,64]. Auranofin has already been shown to primarily inhibit TrxR by irreversible binding to the selenocysteinyl residue, but its exact mode of action is still unclear [61]. In fungi, TrxR was found to be essential for C. neoformans viability and to play a role in responding to oxidative stress in C. albicans, Saccharomyces cerevisiae, and A. fumigatus. Thus, its inhibition by auranofin leads to a higher fungal sensitivity to oxidative stress [59,64]. Scedosporium species are known to possess more than 30 genes encoding putative antioxidant enzymes, especially those encoding TrxRs, which is one reason for their high resistance to antifungal agents [61]. Thus, its inhibition by auranofin might be an important mechanism of action and a promising approach to impair Scedosporium growth.
Iodoquinol is a hydroxyquinoline used to treat Amoeba infections [40,42]. Its mechanism of action is based on chelating ferrous ions essential for microbial metabolism [65]. Nevertheless, little is known about alterations induced in fungal cells. In Sporothrix species, cells treated with iodoquinol presented a ruptured plasma membrane and leakage of intracellular content, suggesting that the cell surface is affected [42]. Scedosporium cell surface was also affected after iodoquinol exposure. In addition, an increase in neutral lipid content and in cell size were observed [42], similar to what we found by SEM analysis where iodoquinol-treated cells presented thicker conidia compared to control.
Besides the direct antifungal effect of new compounds, the study of their interactions with the current antifungal agents is valuable to check the possibility to improve treatment as a combined therapy. Our results demonstrated that auranofin and iodoquinol increased the antifungal activity of caspofungin, fluconazole, and voriconazole. In addition, a synergistic effect was observed between iodoquinol and caspofungin. In C. albicans and C. neoformans, an additive effect of auranofin with fluconazole and amphotericin B has already been demonstrated [62], and an additive interaction has been found with voriconazole in Scedosporium and Lomentospora species [61]. However, these observations were detected only against a limited number of isolates, indicating that more studies are needed to understand how auranofin interacts with the antifungal drugs currently available in clinical settings. Regarding iodoquinol, little is known about its interaction with other antifungal drugs. Coelho and colleagues demonstrated that no effect is observed with itraconazole and terbinafine against fungi causing chromoblastomycosis [41]. On the other hand, topical formulations of iodoquinol exhibited antifungal properties to treat dermatoses caused by C. albicans, Malassezia spp., and dermatophytes and could be used as topical therapy associated with oral antifungals [41,42]. The Bliss independence model is a probabilistic interpretation of drug interactions and is a well-used method to study the synergism or antagonism of two combined drugs [66,67]. Our data revealed that auranofin displays a synergistic effect with all three antifungal drugs tested. Although presenting distinct results compared to FICI analysis, Bliss independence data corroborated what was observed in terms of fungal viability presented in Figure 7. Regarding iodoquinol, its interaction with caspofungin was synergistic using both FICI and Bliss independence analysis. On the other hand, its interaction with azoles was found to be indifference using FICI and antagonistic in Bliss independence model. These data indicate that the definition of synergy is controversial and varies according to which method is used, suggesting that further and deeper studies are needed to clarify the interactions between auranofin and iodoquinol with the antifungal agents currently used in clinical settings.
The cytotoxicity of auranofin and iodoquinol is a key point for the use of these drugs in clinical settings for the treatment of fungal infections. In vitro analyses have already shown that cytotoxicity of auranofin varies significantly depending on the cell model, ranging from 0.15 to 6.38 µm [68][69][70][71][72]. However, since auranofin is a drug already used in humans to treat rheumatoid arthritis, it is important to consider the observations found in clinical trials. In this context, side effects related to the use of auranofin are considered rare and the most frequent is associated with gastrointestinal disorders such as diarrhea [68]. In addition, these side effects are associated with the long-term use of auranofin to treat rheumatoid arthritis, which requires years of drug administration [73,74]. Thus, it is believed that auranofin toxicity would not impair its use against fungal infections, since it requires a short administration.
Regarding iodoquinol, in vitro cytotoxicity also varies significantly in the literature and ranges from 2.5 to more than 125 µm [42,75]. Side effects are also rare and associated with higher doses and prolonged administration, where patients present headaches, nausea, and vomiting [42,65,76].
In summary, screening of the Pathogen Box library allowed for the identification of two new molecules presenting antifungal activity, as well as two promising repurposing compounds (auranofin and iodoquinol), which were evaluated in more detail in the present study. Considering the results that showed their effects against Scedosporium and Lomentospora species and all the data found in the literature, both compounds are potent candidates for more studies on their use to treat fungal infections, alone or in combination with other antifungal agents.