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The efficient inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by the broad-spectrum herbicide glyphosate validates the shikimate pathway as a promising target for developing antimicrobial, fungicidal and herbicidal agents. The last enzyme of this pathway, chorismate synthase (CS), catalyses an unusual reaction, making it an attractive target for novel inhibitors. Therefore, we tested a series of azo-dyes for their inhibitory potential against CS from the pathogenic fungus ( CS) and identified the azo-dye PH011669 that exhibits a dissociation ( ) and 50% inhibitory constant (IC ) of 1.1 ± 0.1 and 10 ± 1 µM, respectively. Molecular docking and MD simulations provided insight into the mode of inhibition, showing that PH011669 binds to the enzyme's active site primarily through electrostatic interactions. Thus, our study is the first to integrate structural and computational methods to guide future efforts towards designing the next generation of CS inhibitors.

The prevalence of invasive fungal infections worldwide has increased in the last decades. The development of specific drugs targeting pathogenic fungi without producing collateral damage to mammalian cells is a daunting pharmacological challenge. Indeed, many of the toxicities and drug interactions observed with contemporary antifungal therapies can be attributed to \"nonselective\" interactions with enzymes or cell membrane systems found in mammalian host cells. A computer-aided screening strategy against the TRR1 protein of Paracoccidioides lutzii is presented here. Initially, a bank of commercially available compounds from Life Chemicals provider was docked to model by virtual screening simulations. The small molecules that interact with the model were ranked and, among the best hits, twelve compounds out of 3,000 commercially-available candidates were selected. These molecules were synthesized for validation and in vitro antifungal activity assays for Paracoccidioides lutzii and P. brasiliensis were performed. From 12 molecules tested, 3 harbor inhibitory activity in antifungal assays against the two pathogenic fungi. Corroborating these findings, the molecules have inhibitory activity against the purified recombinant enzyme TRR1 in biochemical assays. Therefore, a rational combination of molecular modeling simulations and virtual screening of new drugs has provided a cost-effective solution to an early-stage medicinal challenge. These results provide a promising technique to the development of new and innovative drugs.

The search for new compounds with activity against Paracoccidioides , etiologic agents of Paracoccidioidomycosis (PCM), is extremely necessary due to the current scenario of the available therapeutic arsenal. Treatment is restricted to three classes of antifungals with side effects. Curcumin is a polyphenol with antifungal effects that is extracted from Curcuma longa . The present work aimed to evaluate the activity of curcumin in different species of Paracoccidioides and to evaluate the potential molecular targets of curcumin using computational strategies. In addition, interactions with classic antifungals used in the treatment of PCM were evaluated. Curcumin inhibits the growth of Paracoccidioides spp. exerting a fungicidal effect. The combination of curcumin with amphotericin B, co-trimoxazole, and itraconazole showed a synergistic or additive interaction. Molecular targets as superoxide dismutase, catalase, and isocitrate lyase were proposed based on in silico approaches. Curcumin affects the fungal plasma membrane and increases the production of reactive oxygen species. Therefore, curcumin is a good alternative for the treatment of PCM.

To perform virtual screening of compounds based on natural products targeting isocitrate lyase of . Homology modeling and molecular dynamics simulations were applied in order to obtain conformational models for virtual screening. The selected hits were tested against enzymatic activity of ICL of the dimorphic fungus and growth of the spp. The cytotoxicity and selectivity index of the compounds were defined. 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 activity. The compound 4559339 presented minimum inhibitory concentration of 7.3 μg/ml in with fungicidal effect at this concentration. Thus, a new potential antifungal against is proposed.

Regulatory T (Treg) cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J) and susceptible (B10.A) mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+)CD25(+)Foxp3(+) Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+)CD25(+)Foxp3(+) Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+) and CD8(+) T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+) cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the early T cell response in resistant mice and the clearance mechanisms of fungal cells in susceptible mice.

This study aimed to evaluate the activity of 2'-hydroxychalcone-loaded in nanoemulsion (NLS + 2'chalc), the cytotoxic effect and toxicity against and using a zebrafish model. 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 model. NLS + 2'chalc showed fungicidal activity against 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. NLS + 2'chalc is a potential new alternative treatment for paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment.

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. 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. 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. 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.

The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.

Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E(2)) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E(2) inhibits M-to-Y transition. We assessed temporal gene expression in strain Pb01 in the presence or absence of E(2) at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E(2)-regulated clones were sequenced to identify genes and biological function. E(2)-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E(2) belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E(2)-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E(2), whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E(2). This study characterizes the effect of E(2) at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E(2) may be working through signaling genes that regulate dimorphism.