Antiprotozoal Activity of Benzoylthiourea Derivatives against Trypanosoma cruzi: Insights into Mechanism of Action.

For decades, only two nitroheterocyclic drugs have been used as therapeutic agents for Chagas disease. However, these drugs present limited effectiveness during the chronic phase, possess unfavorable pharmacokinetic properties, and induce severe adverse effects, resulting in low treatment adherence. A previous study reported that N-(cyclohexylcarbamothioyl) benzamide (BTU-1), N-(tert-butylcarbamothioyl) benzamide (BTU-2), and (4-bromo-N-(3-nitrophenyl) carbamothioyl benzamide (BTU-3) present selective antiprotozoal activity against all developmental forms of Trypanosoma cruzi Y strain. In this study, we investigated the mechanism of action of these compounds through microscopy and biochemical analyses. Transmission electron microscopy analysis showed nuclear disorganization, changes in the plasma membrane with the appearance of blebs and extracellular arrangements, intense vacuolization, mitochondrial swelling, and formation of myelin-like structures. Biochemical results showed changes in the mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and plasma membrane fluidity. In addition, the formation of autophagic vacuoles was observed. These findings indicate that BTU-1, BTU-2, and BTU-3 induced profound morphological, ultrastructural, and biochemical alterations in epimastigote forms, triggering an autophagic-dependent cell death pathway.

Synergistic antifungal interaction of N-(butylcarbamothioyl) benzamide and amphotericin B against Cryptococcus neoformans.

Introduction: Cryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity. Methods: In the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans. Results: BTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 µg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 µg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties. Conclusion: These results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

Antifungal Combination of Ethyl Acetate Extract of Poincianella pluviosa (DC.) L. P. Queiros Stem Bark With Amphotericin B in Cryptococcus neoformans.

Cryptococcus neoformans is the leading cause of cryptococcosis, an invasive and potentially fatal infectious disease. Therapeutic failures are due to the increase in antifungal resistance, the adverse effects of drugs, and the unavailability of therapeutic regimens in low-income countries, which limit the treatment of cryptococcosis, increasing the morbidity and mortality associated with these infections. Thus, new antifungal drugs and innovative strategies for the cryptococcosis treatment are urgently needed. The aim of the present study was to evaluate the effect of ethyl acetate fraction (EAF) of Poincianella pluviosa stem bark on planktonic and biofilm mode of growth of C. neoformans. Furthermore, the interaction between the EAF and amphotericin B (AmB) was evaluated in vitro and in Galleria mellonella infection model. Minimal inhibitory concentrations (MICs) of EAF ranged from 125.0 to >1,000.0 µg/ml and >1,000.0 µg/ml for planktonic and sessile cells, respectively. The combination between EAF and AmB exhibited a synergistic fungicidal activity toward C. neoformans, with a fractional inhibitory concentration index (FICI) ranging from 0.03 to 0.06 and 0.08 to 0.28 for planktonic and sessile cells, respectively. Microscopy analyses of planktonic C. neoformans cells treated with EAF, alone or combined with AmB, revealed morphological and ultrastructural alterations, including loss of integrity of the cell wall and cell membrane detachment, suggesting leakage of intracellular content, reduction of capsule size, and presence of vacuoles. Moreover, EAF alone or combined with AmB prolonged the survival rate of C. neoformans-infected G. mellonella larvae. These findings indicate that P. pluviosa may be an important source of new compounds that can be used as a fungus-specific adjuvant for the treatment of cryptococcosis.

Membrane dynamics in Leishmania amazonensis and antileishmanial activities of ß-carboline derivatives.

Two ß-carboline compounds, 8i and 6d, demonstrated in vitro antileishmanial activity against Leishmania (L.) amazonensis promastigotes similar to that of miltefosine (MIL). Estimates of the membrane-water partition coefficient (KM/W) and the compound concentrations in the membrane (cm50) and aqueous phase (cw50) for half maximal inhibitory concentration were made. Whereas these biophysical parameters for 6d were not significantly different from those reported for MIL, 8i showed lower affinity for the parasite membrane (lower KM/W) and a lower concentration of the compound in the membrane required to inhibit the growth of the parasite (lower cm50). A 2-hour treatment of Leishmania promastigotes with the compounds 8i and 6d caused membrane rigidity in a concentration-dependent manner, as demonstrated by the electron paramagnetic resonance (EPR) technique and spin label method. This increased rigidity of the membrane was interpreted to be associated with the occurrence of cross-linking of oxidized cytoplasmic proteins to the parasite membrane skeleton. Importantly, the two ß-carboline-oxazoline derivatives showed low hemolytic action, both in experiments with isolated red blood cells or with whole blood, denoting their great Leishmania/erythrocyte selectivity index. Using electron microscopy, changes in the membrane of both the amastigote and promastigote form of the parasite were confirmed, and it was demonstrated that compounds 8i and 6d decreased the number of amastigotes in infected murine macrophages. Furthermore, 8i and 6d were more toxic to the protozoa than to J774A.1 macrophages, with treated promastigotes exhibiting a decrease in cell volume, mitochondrial membrane potential depolarization, accumulation of lipid bodies, increased ROS production and changes in the cell cycle.

Hydrogel Containing Oleoresin From Copaifera officinalis Presents Antibacterial Activity Against Streptococcus agalactiae.

Streptococcus agalactiae or Group B Streptococcus (GBS) remains a leading cause of neonatal infections worldwide; and the maternal vaginal-rectal colonization increases the risk of vertical transmission of GBS to neonates and development of infections. This study reports the in vitro antibacterial effect of the oleoresin from Copaifera officinalis Jacq. L. in natura (copaiba oil) and loaded into carbomer-hydrogel against planktonic and sessile cells of GBS. First, the naturally extracted copaiba oil was tested for the ability to inhibit the growth and metabolic activity of planktonic and sessile GBS cells. The time-kill kinetics showed that copaiba oil exhibited a dose-dependent bactericidal activity against planktonic GBS strains, including those resistant to erythromycin and/or clindamycin [minimal bactericidal concentration (MBC) ranged from 0.06 mg/mL to 0.12 mg/mL]. Copaiba oil did not inhibit the growth of different Lactobacillus species, the indigenous members of the human microbiota. The mass spectral analyses of copaiba oil showed the presence of diterpenes, and the kaurenoic acid appears to be one of the active components of oleoresin from C. officinalis related to antibacterial activity against GBS. Microscopy analyses of planktonic GBS cells treated with copaiba oil revealed morphological and ultrastructural alterations, displaying disruption of the cell wall, damaged cell membrane, decreased electron density of the cytoplasm, presence of intracellular condensed material, and asymmetric septa. Copaiba oil also exhibited antibacterial activity against established biofilms of GBS strains, inhibiting the viability of sessile cells. Low-cost and eco-friendly carbomer-based hydrogels containing copaiba oil (0.5% - CARB-CO 0.5; 1.0% - CARB-CO 1.0) were then developed. However, only CARB-CO 1.0 preserved the antibacterial activity of copaiba oil against GBS strains. This formulation was homogeneous, soft, exhibited a viscoelastic behavior, and showed good biocompatibility with murine vaginal mucosa. Moreover, CARB-CO 1.0 showed a slow and sustained release of the copaiba oil, killing the planktonic and sessile (established biofilm) cells and inhibiting the biofilm formation of GBS on pre-coated abiotic surface. These results indicate that carbomer-based hydrogels may be useful as topical systems for delivery of copaiba oil directly into de vaginal mucosa and controlling S. agalactiae colonization and infection.

Trypanocidal activity of copaiba oil and kaurenoic acid does not depend on macrophage killing machinery.

Activity, mechanisms of action, and toxicity of natural compounds have been investigated in a context in which knowledge on which pathway is activated remains crucial to understand the action mechanism of these bioactive substances when treating an infected host. Herein, we showed an ability of copaiba oil and kaurenoic acid to eliminate Trypanosoma cruzi forms by infected macrophages through other mechanisms in addition to nitric oxide, reactive oxygen species, iron metabolism, and antioxidant defense. Both compounds induced an anti-inflammatory response with an increase in IL-10 and TGF-ß as well as a decrease in IL-12 production. Despite being able to modulate the immune response in host cells, the antimicrobial activity of copaiba oil and kaurenoic acid seems to be a direct action of the compounds on the parasites, causing their death.

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.