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.

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.

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.

Background/Objectives: Leishmaniasis remains a major neglected tropical disease, and current chemotherapeutic options are limited by toxicity and resistance in Leishmania species, including L. amazonensis. Prenylated coumarins have emerged as promising bioactive scaffolds. Altissimacoumarin D and its analogues inhibit fungal efflux pumps associated with resistance. However, their antileishmanial potential and mechanisms of action remain unclear. Here, we evaluated the in vitro, in vivo, and in silico effects of altissimacoumarin D and seven analogues against L. amazonensis. Methods: In vitro assays were performed to identify active compounds and assess toxicity in keratinocytes. In vivo experiments in hamsters evaluated antileishmanial activity and renal and hepatic toxicity. In silico analyses were conducted to investigate the mechanism of action of the substances. Results: In vitro assays showed that ACS47, ACS48, and ACS51 were the most active and safe compounds. In a hamster infection model, daily administration of ACS47 and ACS48 (2.5 mg/kg) significantly reduced parasite burden and lesion size, while maintaining normal renal and hepatic biochemical parameters. Histological analysis correlated reduced lesion size with marked decreases in amastigote density. Based on in silico analysis, spermidine synthase was supported as a plausible molecular target. Conclusions: Collectively, these findings identify ACS47 and ACS48 as promising lead compounds for future antileishmanial drug development.