Repurposing Benzimidazoles against Causative Agents of Chromoblastomycosis: Albendazole Has Superior In Vitro Activity Than Mebendazole and Thiabendazole.

Chromoblastomycosis (CBM) is a neglected human implantation mycosis caused by several dematiaceous fungal species. Currently available therapy is usually associated with physical methods, especially surgery, and with high refractoriness. Therefore, drug discovery for CBM is essential. Drug repositioning is a strategy used to facilitate the discovery of new treatments for several diseases. The aim of this study was to discover substances with antifungal activity against CBM agents from a collection of drugs previously approved for use in human diseases. A screening was performed with the NIH Clinical Collection against Fonsecaea pedrosoi. Ten substances, with clinical applicability in CBM, inhibited fungal growth by at least 60%. The minimum inhibitory concentration (MIC) of these substances was determined against other CBM agents, and the benzimidazoles albendazole, mebendazole and thiabendazole presented the lowest MIC values. The selectivity index, based on MIC and cytotoxicity of these substances, revealed albendazole to be more selective. To investigate a possible synergism of this benzimidazole with itraconazole and terbinafine, the chequerboard method was used. All interactions were classified as indifferent. Our current results suggest that benzimidazoles have repositioning potential against CBM agents. Albendazole seems to be the most promising, since it presented the highest selectivity against all dematiaceous fungi tested.

Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii.

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 µM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 µM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 µM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 µM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.