New Methylcitrate Synthase Inhibitor Induces Proteolysis, Lipid Degradation and Pyruvate Excretion in Paracoccidioides brasiliensis.

BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by the inhalation of conidia of the genus Paracoccidioides. During the infectious process, fungal cells use several carbon sources, leading to the production of propionyl-CoA. The latter is metabolized by the methylcitrate synthase, a key enzyme of the methylcitrate cycle. We identified an inhibitor compound (ZINC08964784) that showed antifungal activity against P. brasiliensis. METHODS: This work aimed to understand the fungal metabolic response of P. brasiliensis cells exposed to ZINC08964784 through a proteomics approach. We used a glucose-free medium supplemented with propionate in order to simulate the environment found by the pathogen during the infection. We performed pyruvate dosage, proteolytic assay, dosage of intracellular lipids and quantification of reactive oxygen species in order to validate the proteomic results. RESULTS: The proteomic analysis indicated that the fungal cells undergo a metabolic shift due to the inhibition of the methylcitrate cycle and the generation of reactive species. Proteolytic enzymes were induced, driving amino acids into degradation for energy production. In addition, glycolysis and the citric acid cycle were down-regulated while ß-oxidation was up-regulated. The accumulation of pyruvate and propionyl-CoA led the cells to a state of oxidative stress in the presence of ZINC08964784. CONCLUSIONS: The inhibition of methylcitrate synthase caused by the compound promoted a metabolic shift in P. brasiliensis damaging energy production and generating oxidative stress. Hence, the compound is a promising alternative for developing new strategies of therapies against paracoccidioidomycosis.

Prospecting for new catechol-O-methyltransferase (COMT) inhibitors as a potential treatment for Parkinson's disease: a study by molecular dynamics and structure-based virtual screening.

Parkinson's disease (PD) is a neurodegenerative, chronic, and progressive disease, common in the elderly. The catechol-O-methyltransferase (COMT) is a monomeric enzyme involved in dopamine (DA) degradation, the neurotransmitter in deficit in patients with PD. The reference treatment of PD consists of levodopa (L-dopa) administration, which is the precursor of DA. The inhibition of COMT is an adjuvant treatment in PD since it keeps DA levels constant. The goal of this study was to identify drug candidates capable of inhibiting COMT for the treatment of PD and identify important fragments of these molecules. Initially, we analyzed the flexibility of COMT and defined its main conformations in solution regarding the absence (system I) and presence of the S-adenosyl-L-methionine (SAM) cofactor (system II) through molecular dynamics (MD) simulations. Two regions in these structures were selected for molecular docking, firstly the entire cavity where the cofactor and substrates are bound and secondly the specific biding region of the enzyme substrates. Based on the conformations of the MD, the virtual screening (VS) was performed against FDA Approved and Zinc Natural Products databases aiming at the selection of the best compounds. Subsequently, the absorption, distribution, metabolization, excretion, and toxicity (ADMET) properties, as well as drug-score and drug-likeness indexes of the most promising compounds were analyzed. After a detailed analysis of the compounds selected by structure-based VS, it was possible to highlight the fragments most frequently involved in their stability: 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, 9H-Benz(c)indole(3,2,1-ij)(1,5)naphthyridin-9-one and (10R,13S)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17dodecahydrocyclopenta[a]phenanthren-3-one. The identification of these potential fragments is essential for the prospection of more specific inhibitors against COMT using the technique of Fragment-based lead discovery (FBLD). Besides, this study allowed us to identify the potential COMT inhibitors through a complete understanding of molecular-level interactions based on the flexibility of this protein.Communicated by Ramaswamy H. Sarma.

Investigation of thiosemicarbazide free or within chitosan nanoparticles in a murine model of vulvovaginal candidiasis.

Vulvovaginal candidiasis is a serious health problem affecting numerous women around the world. Its treatment is based on antifungals which may not provide an effective cure because of the resistance presented by its etiological pathogens Candida spp. Candida albicans is the most prevalent species related to vulvovaginal candidiasis. Here, we evaluated the in vivo antifungal potential of thiosemicarbazide and thiosemicarbazide encapsulated within chitosan nanoparticles in a murine model of vulvovaginal candidiasis. The results demonstrated the antifungal capacity of free or nanoencapsulated thiosemicarbazide within chitosan to reduce the fungal load in the vaginal tissue of infected mice. In addition, histological analyses indicated the absence or a mild to moderate infection in thiosemicarbazide-treated groups. Statistical tests confirmed the existence of significant differences between the treated and the control groups. Therefore, our results suggest a potential application of thiosemicarbazide and encapsulated thiosemicarbazide as an alternative vulvovaginal candidiasis therapy.

Antifungal activity of Copaíba resin oil in solution and nanoemulsion against Paracoccidioides spp.

Paracoccidioidomycosis (PCM) is a disease caused by fungi of the genus Paracoccidioides. The disease is responsible for high rates of premature deaths and socioeconomic repercussions. The limitations of antifungal agents against PCM have motivated the search for new compounds. In our ongoing exploration of Cerrado plants as potential sources of new antifungal agents, we selected Copaifera langsdorffii oil (Copaíba resin oil) in order to explore its bioactive potential and test a formulation to increase oil stability and solubilization employing Pluronic F-127 to obtain the nanoemulsion of the oil. We aim at testing both Copaíba resin oil and its nanoemulsion against four species of the Paracoccidioides genus. We performed cytotoxicity test in Balb/C3T3 cells, hemolytic activity and interaction of Copaíba resin oil and Copaíba resin oil nanoemulsion (CopaPlu) with the antifungal agents such as amphotericin B, co-trimoxazole, and itraconazole. Moreover, the Copaíba resin oil was analyzed by mass spectrometry to identify its chemical profile. Eventually, a new methodology to prepare the nanoemulsion is presented. The Copaíba resin oil and CopaPlu nanoemulsion inhibited Paracoccidioides sp. growth efficiently, and no cytotoxicity or hemolytic effect was observed at minimum inhibitory concentration (MIC). When combined with amphotericin B, Copaíba resin oil and its nanoemulsion showed an additive effect with reduction of MIC values. The Copaíba resin oil and CopaPlu nanoemulsion is a promising antifungal agent against Paracoccidioides.

IR-780-Albumin-Based Nanocarriers Promote Tumor Regression Not Only from Phototherapy but Also by a Nonirradiation Mechanism.

IR-780 iodide is a fluorescent dye with optical properties in the near-infrared region that has applications in tumor detection and photothermal/photodynamic therapy. This multifunctional effect led to the development of theranostic nanoparticles with both IR-780 and chemotherapeutic drugs such as docetaxel, doxorubicin, and lonidamine. In this work, we developed two albumin-based nanoparticles containing near-infrared IR-780 iodide multifunctional dyes, one of them possessing a magnetic core. Molecular docking with AutoDock Vina studies showed that IR-780 binds to bovine serum albumin (BSA) with greater stability at a higher temperature, allowing the protein binding pocket to better fit this dye. The theoretical analysis corroborates the experimental protocols, where an enhancement of IR-780 was found coupled to BSA at 60 °C, even 30 days after preparation, in comparison to 30 °C. In vitro assays monitoring the viability of Ehrlich ascites carcinoma cells revealed the importance of the inorganic magnetic core on the nanocarrier photothermal-cytotoxic effect. Fluorescence molecular tomography measurements of Ehrlich tumor-bearing Swiss mice revealed the biodistribution of the nanocarriers, with marked accumulation in the tumor tissue (≈3% ID). The histopathological analysis demonstrated strong increase in tumoral necrosis areas after 24 and 72 h after treatment, indicating tumor regression. Tumor regression analysis of nonirradiated animals indicate a IR-780 dose-dependent antitumoral effect with survival rates higher than 70% (animals monitored up to 600 days). Furthermore, an in vivo photothermal therapy procedure was performed and tumor regression was also verified. These results show a novel insight for the biomedical application of IR-780-albumin-based nanocarriers, namely cancer therapy, not only by photoinduced therapy but also by a nonirradiation mechanism. Safety studies (acute oral toxicity, cardiovascular evaluation, and histopathological analysis) suggest potential for clinical translation.

Development, characterization, and in vitro-in vivo evaluation of polymeric nanoparticles containing miconazole and farnesol for treatment of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC) is caused mainly by the opportunistic fungus Candida albicans, and its yeast to hyphae transition is considered a major virulence factor. Farnesol is a molecule that inhibits yeast to hyphae transition. The increased incidence of VVC has influenced a need for developing new therapeutic strategies. The objective was to develop a mucoadhesive nanostructured system composed of miconazole and farnesol co-encapsulated within chitosan nanoparticles. The miconazole presented a minimal inhibitory concentration (MIC) of 1 µg/ml against C. albicans. The farnesol was capable of inhibiting yeast to hyphae transition at levels greater or equal to 300 µM. The combination of miconazole and farnesol showed no change in miconazole MIC. Chitosan nanoparticles containing miconazole and farnesol were prepared by ionic gelation and showed favorable characteristics for use on mucous membranes. They showed size variation and polydispersion index (PDI) after 30 days, but the efficiency of drug encapsulation was maintained. Regarding toxicity in cultured fibroblasts (BALB/c 3T3) the nanoparticles were considered nontoxic. The nanoparticles showed antifungal activity against the C. albicans strain used with MICs of 2.5 µg/ml and 2 µg/ml for nanoparticles containing miconazole or miconazole/farnesol, respectively. Nanoparticles containing farnesol inhibited yeast to hyphae transition at concentrations greater than or equal to 240 µM. The in vivo antifungal activity was assessed in the murine model for VVC. The results suggested that chitosan nanoparticles containing miconazole and farnesol were effective at inhibiting fungal proliferation. Additionally, chitosan nanoparticles containing farnesol were capable of decreasing the pathogenicity of infection, demonstrated through the absence of inflammation.

Computer-aided identification of novel anti-paracoccidioidomycosis compounds.

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.

ß-Carboline alkaloids from Galianthe ramosa inhibit malate synthase from Paracoccidioides spp.

As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.

Oenothein B inhibits the expression of PbFKS1 transcript and induces morphological changes in Paracoccidioides brasiliensis.

The fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), the most prevalent human systemic mycosis in Latin America. Drug toxicity and the appearance of resistant strains have created the need to search for new therapeutic approaches. Plants with reputed antimicrobial properties represent a rich screening source of potential antifungal compounds. In this work, the growth of P. brasiliensis yeast cells was evaluated in the presence of oenothein B extracted from Eugenia uniflora. The oenothein B dosage that most effectively inhibited the development (74%) of P. brasiliensis yeast cells in vitro was 500 microg/ml. To verify if oenothein B interferes with cell morphology, we observed oenothein B-treated yeast cells by electron microscopy. The micrographs showed characteristic cell changes noted with glucan synthesis inhibition, including squashing, rough surface, cell wall rupture and cell membrane recess. The expression of P. brasiliensis genes was evaluated in order to investigate the action of oenothein B. Here we report that oenothein B inhibits 1,3-beta-glucan synthase (PbFKS1) transcript accumulation. The results indicate that oenothein B interferes with the cell morphology of P. brasiliensis, probably by inhibiting the transcription of 1,3-beta-glucan synthase gene, which is involved in the cell wall synthesis.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: representational difference analysis identifies candidate genes associated with fungal pathogenesis.

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans.

Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.