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The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

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A series of new metal complexes, [Cu(ITZ)2Cl2] ⋅ 5H2O (1), [Cu(NO3)2(ITZ)2] ⋅ 3H2O ⋅ C4H10O (2) and [Cu(ITZ)2)(PPh3)2]NO3 ⋅ 5H2O (3) were synthesized by a reaction of itraconazole (ITZ) with the respective copper salts under reflux. The metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV-Vis, infrared and EPR spectroscopies. The antifungal activity of these metal complexes was evaluated against the main sporotrichosis agents: Sporothrix brasiliensis, Sporothrix schenkii, and Sporothrix globosa. All three new compounds inhibited the growth of S. brasiliensis and S. schenckii at lower concentrations than the free azole, with complex 2 able to kill all species at 4 µM and induce more pronounced alterations in fungal cells. Complexes 2 and 3 exhibited higher selectivity and no mutagenic effect at the concentration that inhibited fungal growth and affected fungal cells. The strategy of coordinating itraconazole (ITZ) to copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activity of the Cu-ITZ complexes makes them potential candidates for the development of an alternative drug to treat mycoses.

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Mucorales are a group of non-septated filamentous fungi widely distributed in nature, frequently associated with human infections, and are intrinsically resistant to many antifungal drugs. For these reasons, there is an urgent need to improve the clinical management of mucormycosis. Miltefosine, which is a phospholipid analogue of alkylphosphocholine, has been considered a promising repurposing drug to be used to treat fungal infections. In the present study, miltefosine displayed antifungal activity against a variety of Mucorales species, and it was also active against biofilms formed by these fungi. Treatment with miltefosine revealed modifications of cell wall components, neutral lipids, mitochondrial membrane potential, cell morphology, and the induction of oxidative stress. Treated Mucorales cells also presented an increased susceptibility to SDS. Purified ergosterol and glucosylceramide added to the culture medium increased miltefosine MIC, suggesting its interaction with fungal lipids. These data contribute to elucidating the effect of a promising drug repurposed to act against some relevant fungal pathogens that significantly impact public health.

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Candida species are one of the most concerning causative agents of fungal infections in humans. The treatment of invasive Candida infections is based on the use of fluconazole, but the emergence of resistant isolates has been an increasing concern which has led to the study of alternative drugs with antifungal activity. Sphingolipids have been considered a promising target due to their roles in fungal growth and virulence. Inhibitors of the sphingolipid biosynthetic pathway have been described to display antifungal properties, such as myriocin and aureobasidin A, which are active against resistant Candida isolates. In the present study, aureobasidin A did not display antibiofilm activity nor synergism with amphotericin B, but its combination with fluconazole was effective against Candida biofilms and protected the host in an in vivo infection model. Alterations in treated cells revealed increased oxidative stress, reduced mitochondrial membrane potential and chitin content, as well as altered morphology, enhanced DNA leakage and a greater susceptibility to sodium dodecyl sulphate (SDS). In addition, it seems to inhibit the efflux pump CaCdr2p. All these data contribute to elucidating the role of aureobasidin A on fungal cells, especially evidencing its promising use in clinical resistant isolates of Candida species.

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Fungal infections are a global health problem with high mortality and morbidity rates. Available antifungal agents have high toxicity and pharmacodynamic and pharmacokinetic limitations. Moreover, the increased incidence of antifungal-resistant isolates and the emergence of intrinsically resistant species raise concerns about seeking alternatives for efficient antifungal therapy. In this context, we review literature data addressing the potential action of miltefosine (MFS), an anti-Leishmania and anticancer agent, as a repositioning drug for antifungal treatment. Here, we highlight the in vitro and in vivo data, MFS possible mechanisms of action, case reports, and nanocarrier-mediated MFS delivery, focusing on fungal infection therapy. Finally, many studies have demonstrated the promising antifungal action of MFS in vitro, but there is little or no data on antifungal activity in vertebrate animal models and clinical trials, so have a need to develop more research for the repositioning of MFS as an antifungal therapy.

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The subclass naphthoquinone represents a substance group containing several compounds with important activities against various pathogenic microorganisms. Accordingly, we evaluated O-allyl-lawsone (OAL) antiparasitic and antifungal activity free and encapsulated in 2-hydroxypropyl-ß-cyclodextrin (OAL MKN) against Trypanosoma cruzi and Sporothrix spp. OAL and OAL MKN were synthesized and characterized by physicochemical methods. The IC50 values of OAL against T. cruzi were 2.4 µM and 96.8 µM, considering epimastigotes and trypomastigotes, respectively. At the same time, OAL MKN exhibited a lower IC50 value (0.5 µM) for both trypanosome forms and low toxicity for mammalian cells. Additionally, the encapsulation showed a selectivity index approximately 240 times higher than that of benznidazole. Regarding antifungal activity, OAL and OAL MKN inhibited Sporothrix brasiliensis growth at 16 µM, while Sporothrix schenckii was inhibited at 32 µM. OAL MKN also exhibited higher selectivity toward fungus than mammalian cells. In conclusion, we described the encapsulation of O-allyl-lawsone in 2-hydroxypropyl-ß-cyclodextrin, increasing the antiparasitic activity compared with the free form and reducing the cytotoxicity and increasing the selectivity towardSporothrix yeasts and the T. cruzi trypomastigote form. This study highlights the potential development of this inclusion complex as an antiparasitic and antifungal agent to treat neglected diseases.

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Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 µM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 µM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis.

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Mucormycosis is considered concerning invasive fungal infections due to its high mortality rates, difficult diagnosis and limited treatment approaches. Mucorales species are highly resistant to many antifungal agents and the search for alternatives is an urgent need. In the present study, a library with 400 compounds called the Pandemic Response Box® was used and four compounds were identified: alexidine and three non-commercial molecules. These compounds showed anti-biofilm activity, as well as alterations in fungal morphology and cell wall and plasma membrane structure. They also induced oxidative stress and mitochondrial membrane depolarization. In silico analysis revealed promising pharmacological parameters. These results suggest that these four compounds are potent candidates to be considered in future studies for the development of new approaches to treat mucormycosis.

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The increase in the prevalence and severity of fungal infections and the resistance to available antifungals highlights the imperative need for novel therapeutics and the search for new targets. High-content screening of libraries containing hundreds of compounds is a powerful strategy for searching for new drug candidates. In this study, we screened the Pandemic Response Box library (Medicines for Malaria Venture) of 400 diverse molecules against the Sporothrix pathogenic species. The initial screen identified twenty-four candidates that inhibited the growth of Sporothrix brasiliensis by more than 80%. Some of these compounds are known to display antifungal activity, including olorofim (MMV1782354), a new antifungal drug. Olorofim inhibited and killed the yeasts of S. brasiliensis, S. schenckii, and S. globosa at concentrations lower than itraconazole, and it also showed antibiofilm activity. According to the results obtained by fluorimetry, electron microscopy, and particle characterization analyses, we observed that olorofim induced profound alterations on the cell surface and cell cycle arrest in S. brasiliensis yeasts. We also verified that these morphophysiological alterations impaired their ability to adhere to keratinocytes in vitro. Our results indicate that olorofim is a promising new antifungal against sporotrichosis agents.

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Sporotrichosis, a human and animal disease caused by Sporothrix species, is the most important implantation mycosis worldwide. Sporothrix taxonomy has improved in recent years, allowing important advances in diagnosis, epidemiology, and treatment. Molecular epidemiology reveals that S. brasiliensis remains highly prevalent during the cat-transmitted sporotrichosis outbreaks in South America and that the spread of S. brasiliensis occurs through founder effects. Sporothrix globosa and S. schenckii are cosmopolitan on the move, causing major sapronoses in Asia and the Americas, respectively. In this emerging scenario, one-health approaches are required to develop a creative, effective, and sustainable response to tackle the spread of sporotrichosis. In the 21st century, it has become vital to speciate Sporothrix, and PCR is the main pillar of molecular diagnosis, aiming at the detection of the pathogen DNA from clinical samples through multiplex assays, whose sensitivity reaches remarkably three copies of the target. The treatment of sporotrichosis can be challenging, especially after the emergence of resistance to azoles and polyenes. Alternative drugs arising from discoveries or repositioning have entered the radar of basic research over the last decade and point to several molecules with antifungal potential, especially the hydrazone derivatives with great in vitro and in vivo activities. There are many promising developments for the near future, and in this review, we discuss how these trends can be applied to the Sporothrix-sporotrichosis system to mitigate the advance of an emerging and re-emerging disease.

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Sporotrichosis is a subcutaneous mycosis that affects humans and animals, with few therapeutic options available in the pharmaceutical market. We screened the in vitro antifungal activity of fourteen 1,4-naphthoquinones derivative compounds against Sporothrix brasiliensis and Sporothrix schenckii, the main etiological agents of sporotrichosis in Latin America. The most active compound was selected for further studies exploring its antibiofilm activity, effects on yeast morphophysiology, interaction with itraconazole, and selectivity to fungal cells. Among the fourteen 1,4-naphthoquinones tested, naphthoquinone 5, a silver salt of lawsone, was the most active compound. Naphthoquinone 5 was able to inhibit Sporothrix biofilms and induced ROS accumulation, mitochondrial disturbances, and severe plasmatic membrane damage in fungal cells. Furthermore, naphthoquinone 5 was ten times more selective towards fungal cells than fibroblast, and the combination of itraconazole with naphthoquinone 5 improved the inhibitory activity of the azole. Combined, the data presented here indicate that the silver salt naphthoquinone 5 exerts promising in vitro activity against the two main agents of sporotrichosis with important antibiofilm activity and a good toxicity profile, suggesting it is a promising molecule for the development of a new family of antifungals.

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Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp. The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Psd2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Psd2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Psd2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 µM Psd2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Psd2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Psd2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.

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Sporotrichosis is the most prevalent subcutaneous mycosis globally, and it is typically caused by direct inoculation of the soil saprophytic fungus Sporothrix spp. into the patients' skin. However, sporotrichosis has an important zoonotic transmission route between cats and humans in hot-spot endemic areas such as Brazil. Antifungal itraconazole is the first-line treatment; however, it is frequently associated with recurrence after withdrawal, mainly on cats. Biofilms are important resistance structures related to the environmental persistence of most microorganisms. In the present work, we evaluated Sporothrix yeasts' ability to form biofilms in an ex vivo model of infected claws of cats. Using scanning electron microscopy, we demonstrated the presence of fungal biofilms in the claws of cats diagnosed with sporotrichosis confirmed by isolation of Sporothrix spp. in culture. We present here evidence of antibiofilm activity of miltefosine and suggest its use off-label as an antifungal as a putative alternative to itraconazole against Sporothrix biofilms. Claw contamination could sustain infections through a continuous inoculation cycle between open lesions and cat claws. Our results further support the off-label use of miltefosine as a promising alternative, especially for mycosis refractory to conventional treatment.

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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.

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Scedosporium and Lomentospora species are filamentous fungi responsible for a wide range of infections in humans and are frequently associated with cystic fibrosis and immunocompromising conditions. Because they are usually resistant to many antifungal drugs available in clinical settings, studies of alternative targets in fungal cells and therapeutic approaches are necessary. In the present work, we evaluated the in vitro antifungal activity of miltefosine against Scedosporium and Lomentospora species and how this phospholipid analogue affects the fungal cell. Miltefosine inhibited different Scedosporium and Lomentospora species at 2-4 µg/ml and reduced biofilm formation. The loss of membrane integrity in Scedosporium aurantiacum caused by miltefosine was demonstrated by leakage of intracellular components and lipid raft disorganisation. The exogenous addition of glucosylceramide decreased the inhibitory activity of miltefosine. Reactive oxygen species production and mitochondrial activity were also affected by miltefosine, as well as the susceptibility to fluconazole, caspofungin and myoricin. The data obtained in the present study contribute to clarify the dynamics of the interaction between miltefosine and Scedosporium and Lomentospora cells, highlighting its potential use as new antifungal drug in the future.

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Sporotrichosis has become an important zoonosis in Brazil, and Sporothrix brasiliensis is the primary species transmitted by cats. Improvement of animal treatment will help control and limit the spread and geographic expansion of sporotrichosis. Accordingly, buparvaquone, an antiprotozoal hydroxynaphthoquinone agent marketed as Butalex, was evaluated in vitro and in vivo against feline-borne isolates of S. brasiliensis. Buparvaquone inhibited in vitro fungal growth at concentrations 4-fold lower than itraconazole (the first-choice antifungal used for sporotrichosis) and was 408 times more selective for S. brasiliensis than mammalian cells. Yeasts treated with a subinhibitory concentration of buparvaquone exhibited mitochondrial dysfunction, reactive oxygen species and neutral lipid accumulation, and impaired plasma membranes. Scanning electron microscopy images also revealed buparvaquone altered cell wall integrity and induced cell disruption. In vivo experiments in a Galleria mellonella model revealed that buparvaquone (single dose of 5 mg/kg of body weight) is more effective than itraconazole against infections with S. brasiliensis yeasts. Combined, our results indicate that buparvaquone has a great in vitro and in vivo antifungal activity against S. brasiliensis, revealing the potential application of this drug as an alternative treatment for feline sporotrichosis.

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A series of new metal complexes, [Zn(KTZ)2(Ac)2]·H2O (1), [Zn(KTZ)2Cl2]·0.4CH3OH (2), [Zn(KTZ)2(H2O)(NO3)](NO3) (3), [Cu(KTZ)2(Ac)2]·H2O (4), [Cu(KTZ)2Cl2]·3.2H2O (5), [Cu(KTZ)2(H2O)(NO3)](NO3)·H2O (6), were synthesized by a reaction of ketoconazole (KTZ) with their respective zinc or copper salts under mild conditions. Similarly, six corresponding metal-CTZ (clotrimazole) complexes [Zn(CTZ)2(Ac)2]·4H2O (7), [Zn(CTZ)2Cl2] (8), [Zn(CTZ)2(H2O)(NO3)](NO3)·4H2O (9), [Cu(CTZ)2(Ac)2]·H2O (10), [Cu(CTZ)2Cl2]·2H2O (11), [Cu(CTZ)2(H2O)(NO3)](NO3)·2H2O (12), were obtained. These metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV/Vis, and infrared spectroscopies. Further, the crystal structure for complexes 7 and 10 was determined by single-crystal X-ray diffraction. The antifungal activity of these metal complexes was evaluated against three fungal species of medical relevance: Candida albicans, Cryptococcus neoformans, and Sporothrix brasiliensis. Complexes 1 and 3 exhibited the greatest antifungal activity with a broad spectrum of action at low concentrations and high selectivity. Some morphological changes induced by these metal complexes in S. brasiliensis cells included yeast-hyphae conversion, an increase in cell size and cell wall damage. The strategy of coordination of clinic drugs (KTZ and CTZ) to zinc and copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activities displayed by Zn-KTZ complexes make them potential candidates for the development of an alternative drug to treat mycoses.

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The in vitro activity of ibuprofen, a nonsteroidal anti-inflammatory drug, was evaluated against Sporothrix brasiliensis and S. schenckii, either alone or in combination with amphotericin B, itraconazole, or terbinafine. The inhibitory activity of ibuprofen as a single agent was determined according to minimum inhibitory concentration (MIC) values, while the effect of ibuprofen combined with amphotericin B, itraconazole, or terbinafine was estimated by microdilution checkerboard methodology. The ultrastructural alterations of S. schenckii after exposure to the combination of ibuprofen and amphotericin B were evaluated by scanning electron microscopy (SEM) and flow cytometry analysis. As a single agent, ibuprofen inhibited Sporothrix growth with a MIC median of 256 µg/mL, while the MIC medians of ibuprofen in combination with antifungals were 16 µg/mL and 128 µg/mL. The MIC values of amphotericin B, itraconazole, and terbinafine were reduced when isolates were co-incubated with ibuprofen, mainly the polyene. The major alteration after treatment with the ibuprofen/amphotericin B combination was the increase in the presence of filamentous forms and high membrane damage with loss of plasma membrane integrity. In summary, we demonstrated that ibuprofen increases the in vitro activity of antifungals, mainly amphotericin B, against S. brasiliensis and S. schenckii. Future in vivo studies exploring combination therapy with ibuprofen and antifungals in animal models are needed to confirm its efficacy.

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Sporotrichosis is a neglected endemic mycosis with a high incidence in Latin America, mainly in Brazil. Sporothrix schenckii is the most frequent species in Latin America, whereas Sporothrix brasiliensis is the predominant species observed in Brazil and is associated with both human and animal sporotrichosis. Sporotrichosis treatment remains restricted to a few options, itraconazole being the first choice for human and animal therapy. In this work, we screened the molecular library Pathogen Box (Medicines for Malaria Venture [MMV], Switzerland) in search of compounds with anti-Sporothrix activity. Our initial screen of the 400 compounds identified five compounds that inhibited more than 80% of S. brasiliensis and S. schenkii growth. Among those, three compounds (MMV675968, MMV102872, and MMV002817 (known as iodoquinol)) not previously described as antifungals or agrochemicals, were selected for further evaluation. MMV102872 and iodoquinol showed the most promising combination of antifungal activity (lower inhibitory concentration) and fungal selectivity (lower cytotoxicity in LLC-MK2 cells). Scanning electron microscopy and flow cytometry analyses revealed that MMV102872 and iodoquinol induced changes in cell morphology, membrane integrity, and the presence of neutral lipids, impairing fungal survival. Our results indicate that MMV102872 and iodoquinol are promising molecules for use as scaffolds for the development of new antifungal agents.

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In recent years, the development of new pharmaceutical formulations for the treatment of sporotrichosis has become a relevant research field. In this work, we aimed to develop an emulgel containing itraconazole and clotrimazole to ensure therapeutic effectiveness against Sporothrix brasiliensis. The topical use of a formulation that combines both drugs represents an interesting option for the complementary treatment of sporotrichosis. The emulgel formulation was prepared and evaluated for its zeta potential, viscosity, in vitro antifungal activity and stability at different storage conditions. The results showed that the newly developed emulgel displayed promising physicochemical characteristics, as well as a good in vitro inhibitory activity against S. brasiliensis yeasts. The results obtained in this work suggest that the emulgel containing itraconazole and clotrimazole might highly be efficient and a complementary therapy to oral administration in the treatment of sporotrichosis.

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