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Aim: To search for potential inhibitors to homoserine dehydrogenase (HSD) in Paracoccidioides brasiliensis the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil. Materials & methods: The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs. Results: The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of Pb HSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 μg/ml and MFC of 64 μg/ml against the P. brasiliensis (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines. Conclusion: These results suggest that the HS23 and HS87 are promising candidates as Pb HSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis.

Aim: This study aimed to evaluate the activity of 2'-hydroxychalcone-loaded in nanoemulsion (NLS + 2'chalc), the cytotoxic effect and toxicity against Paracoccidioides brasiliensis and Paracoccidioides lutzii using a zebrafish model. Materials & methods: Preparation and physical-chemical characterization of nanoemulsion (NLS) and NLS + 2'chalc were performed. MIC and minimum fungicide concentration, cytotoxicity and toxicity were also evaluated in the Danio rerio model. Results: NLS + 2'chalc showed fungicidal activity against Paracoccidioides spp. without cytotoxicity in MRC5 and HepG2 lines. It also had high selectivity index values and no toxicity in the zebrafish model based on MIC values. Conclusion: NLS + 2'chalc is a potential new alternative treatment for paracoccidioidomycosis.

Aim: To perform virtual screening of compounds based on natural products targeting isocitrate lyase of Paracoccidioides brasiliensis . Materials & methods: Homology modeling and molecular dynamics simulations were applied in order to obtain conformational models for virtual screening. The selected hits were tested in vitro against enzymatic activity of ICL of the dimorphic fungus P. brasiliensis and growth of the Paracoccidioides spp. The cytotoxicity and selectivity index of the compounds were defined. Results & conclusion: Carboxamide, lactone and β-carboline moieties were identified as interesting chemical groups for the design of new antifungal compounds. The compounds inhibited ICL of the dimorphic fungus P. brasiliensis activity. The compound 4559339 presented minimum inhibitory concentration of 7.3 μg/ml in P. brasiliensis with fungicidal effect at this concentration. Thus, a new potential antifungal against P. brasiliensis is proposed.

Aim: A structural model of chorismate synthase (CS) from the pathogenic fungus Candida albicans was used for virtual screening simulations. Methods: Docking, molecular dynamics, cell growth inhibition and protein binding assays were used for search and validation. Results: Two molecules termed CS8 and CaCS02 were identified. Further studies of the minimal inhibitory concentration demonstrated fungicidal activity against Paracoccidioides brasiliensis with a minimal inhibitory concentration and minimal fungicidal concentration of 512 and 32 μg·ml -1 for CS8 and CaCS02, respectively. In addition, CaCS02 showed a strong synergistic effect in combination with amphotericin B without cytotoxic effects. In vitro studies using recombinant CS from P. brasiliensis showed IC 50 of 29 μM for CaCS02 supporting our interpretation that inhibition of CS causes the observed fungicidal activity.

Aim:  17 new 4-methoxynaphthalene- N -acylhydrazones were synthesized in order to evaluate their biological action against important pathogens. Methods:   In vitro susceptibility assays of compounds were performed against Paracoccidioides brasiliensis and Mycobacterium tuberculosis . Results: Compounds 4a , 4b and 4k were the most potent against P. brasiliensis , two with minimum inhibitory concentrations of ≤1 μg ml -1 and exhibited pharmacological synergy with amphotericin B. The compounds also showed activity against M. tuberculosis , with 4c and 4k being the more promising. Compound 4k showed good synergistic antimycobacterium activity with ethambutol. None of the compounds tested showed toxicity. Conclusion: We highlight the compound 4k , as a potential agent for the treatment of patients co-infected with paracoccidioidomycosis and tuberculosis.

Aim: Novel 4-methoxy-naphthalene derivatives were synthesized based on hits structures in order to evaluate the antifungal activity against Paracoccidioides spp., Methods: Antifungal activity of compounds was evaluated against P. brasiliensis and most promising compounds 2 and 3 were tested against eight clinically important fungal species., Results: Compound 3 was the more active compound with MIC 8 to 32 μg.ml -1 for Paracoccidioides spp without toxicity monkey kidney and murine macrophagecells. Carbohydrazide 3 showed good synergistic antifungal activity with amphotericin B against P. brasiliensis specie. Titration assay of carbohydrazide 3 with PbHSD enzyme demonstrates the binding ligand-protein. Molecular dynamics simulations show that ligand 3 let the PbHSD protein more stable., Conclusion: New carbohydrazide 3 is an attractive lead for drug development to treat paracoccidioidomycoses.

Aim: The shape-based virtual screening was used for the identification of new compounds anti-paracoccidioidomycosis (PCM)., Materials & Methods: The study was performed according to the following steps: collection and curation of a dataset of quinolinyl N-oxide chalcones with anti-PCM activity, development and validation of shape-based models, application of the best model for virtual screening, and experimental validation., Results & Conclusion: Among 31 computational hits, eight compounds showed potent antifungal activity and low cytotoxicity for mammalian cells. The checkerboard assay showed that most promising hit (compound 3) displayed additive effects with the antifungal cotrimoxazole and amphotericin B. Therefore, the shape-based virtual screening allowed us to discover promising compounds in prospective hit-to-lead optimization studies for tackling PCM.

Aim: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action., Methods: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound., Results: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors., Conclusion: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.