In vitro antifungal activity of MMV Pathogen Box® compounds alone or in combination with antifungal drugs against mucormycosis agents.

Mucormycosis is a severe fungal infection that demands immediate and decisive intervention upon suspicion. The causative agents of mucormycosis exhibit inherent resistance to echinocandins and voriconazole, and their in vitro susceptibility to terbinafine is highly variable and species-specific. Considering these factors and the limitations of currently available antifungal therapies, the identification of novel antifungals with potent activity against mucormycosis is of paramount importance. This study aims to identify compounds from the MMV Pathogen Box® presenting antifungal activity against selected mucormycosis agents and to evaluate their potential synergistic effects when combined with antifungal drugs. A screening of the Pathogen Box® compounds was conducted, isolated or in combination with sub-inhibitory concentrations of amphotericin B, isavuconazole or posaconazole, against a Rhizopus oryzae strain. Hits from the screenings were further evaluated against eight Mucoralean strains for minimal inhibitory and fungicidal concentration determinations and to confirm synergistic interactions using the checkerboard method. Ultrastructural studies were performed using scanning electron microscopy. MMV675968 exhibited fungicidal activity against a R. oryzae strain. All but one Rhizopus spp. strains presented MIC ≤ 1 µg/mL, with a geometric mean of 0.78 µg/mL observed across all isolates for this compound, which did not change significantly the cellular structure of this fungus. The combination screening with antifungal drugs revealed six additional compounds potentially active against the R. oryzae strain, two of them demonstrated proven synergism through the checkerboard assay. This first study with the MMV Pathogen Box® and Zigomycetes highlights promising new treatment options for mucormycosis in the future.

Assessing the In Vitro Potential of Glatiramer Acetate (Copaxone®) as a Chemotherapeutic Candidate for the Treatment of Cryptococcus neoformans Infection.

Cryptococcosis is a systemic mycosis affecting immunosuppressed individuals, caused by various Cryptococcus species. The current treatment utilizes a combination of antifungal drugs, but issues such as nephrotoxicity, restricted or limited availability in certain countries, and resistance limit their effectiveness. Repurposing approved drugs presents a viable strategy for developing new antifungal options. This study investigates the potential of glatiramer acetate (Copaxone®) as a chemotherapy candidate for Cryptococcus neoformans infection. Various techniques are employed to evaluate the effects of glatiramer acetate on the fungus, including microdilution, XTT analysis, electron and light microscopy, and physicochemical measurements. The results demonstrate that glatiramer acetate exhibits antifungal properties, with an IC50 of 0.470 mg/mL and a minimum inhibitory concentration (MIC) of 2.5 mg/mL. Furthermore, it promotes enhanced cell aggregation, facilitates biofilm formation, and increases the secretion of fungal polysaccharides. These findings indicate that glatiramer acetate not only shows an antifungal effect but also modulates the key virulence factor-the polysaccharide capsule. In summary, repurposing glatiramer acetate as a potential chemotherapy option offers new prospects for combating C. neoformans infection. It addresses the limitations associated with current antifungal therapies by providing an alternative treatment approach.

Screening of Pandemic Response Box Library Reveals the High Activity of Olorofim against Pathogenic Sporothrix Species.

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.

Extracellular Vesicles Regulate Biofilm Formation and Yeast-to-Hypha Differentiation in Candida albicans.

In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.