Establishment of a three-dimensional model to determine the pharmacological efficacy and safety of nitrofuran and indoles derivatives and evaluation of Histoplasma capsulatum infection

Three-dimensional (3D) cultures are a type of cell culture that are intended to simulate tissue in vivo more faithfully. Fungal diseases affect more than 1 billion people worldwide. The increasing evolution of microbial resistance and the adverse effects of current antifungal drugs assist in the emergence of new drugs. This study aims to establish lung cell spheroids to evaluate the toxicity of nitrofuran and indole derivatives and explore the H. capsulatum-host interaction. Furthermore, we aim to study the toxicity of these compounds in vivo using alternative animals, as well as to investigate their possible mechanism(s) of action. Spheroids from the A549 and MRC-5 lung lines were characterized by Trypan blue, resazurin and propidium iodide methods. Cell size, shape and density were evaluated by scanning electron microscopy (SEM) and confocal microscopy. Sensitivity tests were conducted according to document M27-A3, proposed by the Clinical and Laboratory Standards Institute (CLSI), with minor modifications in the assays with H. capsulatum. Cytotoxicity was assessed in spheroids and in monolayer by addition of resazurin. In vivo toxicity was evaluated in the Caenorhabditis elegans, Galleria mellonella and Zebrafish model. Antibiofilm action of the most potent compounds was verified for H. capsulatum by the tetrazolium salt reduction (XTT) method. The damage in the mature biofilms was visualized by SEM and confocal. The mechanism of action was ascertained by quantification of membrane ergosterol, verification of wall damage with Calcofluor White, production of reactive oxygen species (ROS) using 2,7-dichlorodihydrofluorescein, cell death by propidium iodide (PI)/Anexin assay and real- time PCR technique. Interactions between planktonic and mature biofilm cells of H. capsulatum were performed on the spheroids and compared to infections under the same conditions in monolayers by real-time PCR. The spheroids showed diameters between 370- 600 μm and viability higher than 80% after 10 days of formation. SEM and confocal showed an intact spheroidal architecture consisting of cells interconnected with each other. Of the compounds tested against H. capsulatum, 18 were most effective with MIC90 values from 0.122 to 61.25 μg/mL. Overall, the compounds tested were less toxic on spheroids compared to the monolayer. Among the compounds tested L7CF 166 (nitrofuran derivative), L7CF 197 (indole derivative) and L7CF 219 (nitrofuran/indole derivative) were the most specific for H. capsulatum. In the alternative models C. elegans, G. mellonella and zebrafish the toxicity of the compounds was low, even at high concentrations. The compounds inhibited the initial and mature biofilm at concentration equal to or twice that required for planktonic cells. Micrographs showed a reduction in metabolic activity, thickness, and extracellular matrix of the biofilms. All three compounds reduced the amount of ergosterol present in the fungal membrane. Two compounds L7CF 166 and L7CF 219 were able to cause deformations in the cell wall. L7CF 197 and L7CF 219, on the other hand, were able to induce ROS in the cells. In addition, all three compounds caused cell death via necrotic pathway. The spheroids infected with the planktonic form obtained a negative modulation for the CBP1 gene. The spheroids infected with biofilm showed positive modulation for CBP1 gene and GAPDH, and negative modulation for DDR8 gene. Additionally, the series of nitrofuran derivatives were tested against different fungal species. Cytotoxicity was evaluated using monolayer and spheroid cultures of A549 and MRC-5 and in vivo toxicity in the alternative C.elegans model. Most of the compounds showed low inhibitory activity. The most potent MIC90 values were 0.48 μg/mL against Paracoccidioides brasiliensis (L7CF 182), 0.98 μg/mL against Trichophyton rubrum and T. mentagrophytes (L7CF 193) and 3.9 μg/mL against Candida and Cryptococcus neoformans strains (L7CF 113 and L7CF 181, respectively). Furthermore, all compounds showed low toxicity when tested in vitro and in vivo. Based on these results, the spheroids show to be viable for drug toxicity study and for infection experiments. Overall, the results suggest that nitrofuran and indole derivatives are promising compounds for the treatment of fungal infections. (AU)