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

Abstract

Histoplasma capsulatum is a fungus that causes Histoplasmosis, a systemic and endemic Mycosis worldwide. In Brazil, this Mycosis mainly affects the southeast region, causing real problems in public health. There is a limited number of antifungal drugs for the treatment of mycoses in general and, particularly of Histoplasmosis, which have significant adverse effects such as renal and hepatic toxicity. This factor added to the growing reports of antifungal resistance and the formation of biofilms by these fungi, makes the development of new antifungals more effective both against planktonic cells and form biofilms, which have reduced toxicity. Therefore, this work aims to verify the anti-Histoplasma activity of nitrofuran and indole derivatives, as well as to investigate its possible mechanism (s) of action. Also, it is intended to establish a three-dimensional model formed by lung cells for the study of the H. capsulatum-host interaction using molecular methodologies and to evaluate the toxicity of the compounds. Two strains of H. capsulatum, EH-315 and ATCC 26032 (G217-B) will be used. The compounds derived from nitrofurans and indoles were elaborated and produced by the research group of Prof. Dr. Luigi Scipione from the University of Rome, Italy. The sensitivity tests will be conducted according to document M27-A3, proposed by the Clinical and Laboratory Standards Institute (CLSI, 2008), with minor modifications. The minimum fungicidal concentration (CFM) will be performed. The most potent compounds will be subjected to cytotoxicity testing in three-dimensional and monolayer cultures of cell lines A549 and MRC-5. The compounds that obtain the best selectivity indexes will be evaluated for their ability to inhibit planktonic cells in the concentration of biofilm, initial biofilm, and mature biofilm. For this, the metabolic activities of biofilms (planktonic biofilm concentration, initial and mature) will be quantified by the method of reducing the tetrazolic salt XTT and Scanning Electron Microscopy (SEM) images will be obtained, to verify possible damage. To determine the mechanisms of action will be performed: the quantification of the membrane ergosterol, evaluation of the cell death mechanism by the Propidium Iodide (PI) and annexin (FITC) assay, the dosage of Reactive Oxygen Species (ROS), wall damage, and cell membrane by SEM, confocal microscopy and transmission microscopy. In addition, toxicity will be verified in larvae of Caenorhabditis elegans, teratogenicity in Zebrafish embryos, and mutagenicity by micronucleus test. (AU)