Search Results (46)

Background/Objectives: α-carbonic anhydrase (α-TcCA) has emerged as a promising drug target in T. cruzi, the causative agent of Chagas disease in the Americas. Sulfonamides, known inhibitors of CAs, bind to the zinc ion on the enzyme’s active site. This study proposes the repositioning of sulfonamide-based drugs to identify new trypanocidal agents. Method: Ligand-based virtual screening and molecular docking analysis were performed on FDA-approved drugs targeting α-TcCA. These compounds were evaluated in vitro and ex vivo against the A1 and NINOA strains, followed by enzymatic assays. Results: Four sulfonylureas were selected: glimepiride (Glim), acetohexamide (Ace), gliclazide (Glic), and tolbutamide (Tol). Ace and Tol had half-maximal inhibitory concentration (IC₅₀) values similar or better than reference drugs against the NINOA strain in the epimastigote and trypomastigote stages, while Glic and Glim had the highest activity against the A1 strain (epimastigotes and amastigotes). Notably, Ace had the highest trypanocidal activity against all stages in NINOA, with IC₅₀ values of 6.5, 46.5, and 46 μM for epimastigotes, trypomastigotes, and amastigotes, respectively. Additionally, Ace inhibited α-TcCA with K_I = 5.6 μM, suggesting that its trypanocidal effect is associated to the enzyme inhibition. Conclusions: This study supports the repositioning of FDA-approved sulfonamide-based hypoglycaemic agents as trypanocidal compounds. Future studies should focus on structural modifications to improve selectivity. Integrating docking, parasitological, and enzymatic data is crucial for optimizing drug candidates for Chagas disease. Full article

Background/Objectives: Leishmaniasis is a neglected tropical disease caused by a protozoan parasite of Leishmania. This study aimed to evaluate the in vitro efficacy and toxicity of a previously developed amphotericin gel as a possible treatment for cutaneous leishmaniasis. Methods: First, quality control of the AmB-gel was carried out, including microbiological stability. The permeated and retained drug was tested on healthy and lacerated human skin. Tolerance to the AmB-gel was tested in vitro using HaCaT, RAW 264.7, and J774 cell lines and by an irritation test (HET-CAM). Promastigotes and amastigotes of various Leishmania species were tested, and the microscopic morphology of promastigotes exposed to the formulation was analyzed. Computational analysis was performed on the drug, polymer, and ergosterol in the promastigote. Results: The AmB-gel presented appropriate characteristics for topical use, including no microbial contamination after storage. The amount of drug retained on the intact and injured skin was 1180.00 ± 13.54 µg/g/cm² and 750.18 ± 5.43 µg/g/cm², respectively. The AmB-gel did not cause significant signs of toxicity. The IC₅₀ of the AmB-gel for promastigotes was less than 1 µg/mL for the four species examined, i.e., Leishmania infantum, Leishmania tropica, Leishmania major, and Leishmania braziliensis, and less than 2 µg/mL for amastigotes of Leishmania infantum and Leishmania tropica. The AmB-gel caused notable effects on the surface of promastigotes. Computational analysis revealed primarily hydrophobic and van der Waals interactions between AmB and Pluronic^® F127 and ergosterol. Conclusions: Based on the drug retention content and IC₅₀ values observed for both parasite stages, the AmB-gel may be a promising candidate for in vivo studies in patients with cutaneous leishmaniasis. Full article

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects millions globally, with limited treatment options available. Current therapies, such as benznidazole and nifurtimox, present challenges, including their toxicity, side effects, and inefficacy in the chronic phase. This study explores the potential of drug repurposing as a strategy to identify new treatments for T. cruzi, focusing on compounds from the Medicines for Malaria Venture (MMV) COVID Box. An initial screening of 160 compounds identified eight with trypanocidal activity, with almitrine and bortezomib showing the highest efficacy. Both compounds demonstrated significant activity against the epimastigote and amastigote stages of the parasite and showed no cytotoxicity in murine macrophage cells. Key features of programmed cell death (PCD), such as chromatin condensation, mitochondrial membrane potential disruption, and reactive oxygen species accumulation, were observed in T. cruzi treated with these compounds. The potential to induce controlled cell death of these two compounds in T. cruzi suggests they are promising candidates for further research. This study reinforces drug repurposing as a viable approach to discovering novel treatments for neglected tropical diseases like Chagas disease. Full article

Leishmaniasis, a neglected tropical disease caused by Leishmania species, presents serious public health challenges due to limited treatment options, toxicity, high costs, and drug resistance. In this study, the in vitro potential of malvidin and echioidinin is examined as antileishmanial agents against L. amazonensis, L. braziliensis, and L. infantum, comparing their effects to amphotericin B (AmpB), a standard drug. Malvidin demonstrated greater potency than echioidinin across all parasite stages and species. Against L. amazonensis, malvidin’s IC₅₀ values were 197.71 ± 17.20 µM (stationary amastigotes) and 258.07 ± 17 µM (axenic amastigotes), compared to echioidinin’s 272.99 ± 29.90 μM and 335.96 ± 19.35 μM. AmpB was more potent, with IC₅₀ values of 0.06 ± 0.01 µM and 0.10 ± 0.03 µM. Malvidin exhibited lower cytotoxicity (CC₅₀: 2920.31 ± 80.29 µM) than AmpB (1.06 ± 0.12 µM) and a favorable selectivity index. It reduced infection rates by 35.75% in L. amazonensis-infected macrophages. The in silico analysis revealed strong binding between malvidin and Leishmania arginase, with the residues HIS139 and PRO258 playing key roles. Gene expression analysis indicated malvidin’s modulation of oxidative stress and DNA repair pathways, involving genes like GLO1 and APEX1. These findings suggest malvidin’s potential as a safe, natural antileishmanial compound, warranting further in vivo studies to confirm its therapeutic efficacy and pharmacokinetics in animal models. Full article

Sterol biosynthesis requires the oxidative removal of two methyl groups from the C-4 position by sterol C-4-demethylase and one methyl group from the C-14 position by sterol C-14-demethylase. In Leishmania donovani, a CYP5122A1 (Cytochrome P450 family 5122A1) protein was recently identified as the bona fide sterol C-4 methyl oxidase catalyzing the initial steps of C-4-demethylation. Besides CYP5122A1, Leishmania parasites possess orthologs to ERG25 (ergosterol pathway gene 25), the canonical sterol C-4 methyl oxidase in Saccharomyces cerevisiae. To determine the contribution of CYP5122A1 and ERG25 in sterol biosynthesis, we assessed the essentiality of these genes in Leishmania major, which causes cutaneous leishmaniasis. Like in L. donovani, CYP5122A1 in L. major could only be deleted in the presence of a complementing episome. Even with strong negative selection, L. major chromosomal CYP5122A1-null mutants retained the complementing episome in both promastigote and amastigote stages, demonstrating its essentiality. In contrast, the L. major ERG25-null mutants were fully viable and replicative in culture and virulent in mice. Deletion and overexpression of ERG25 did not affect the sterol composition, indicating that ERG25 is not required for C-4-demethylation. These findings suggest that CYP5122A1 is the dominant and possibly only sterol C-4 methyl oxidase in Leishmania, and inhibitors of CYP5122A1 may have strong therapeutic potential against multiple Leishmania species. Full article

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, also called American trypanosomiasis. This neglected tropical disease affects millions of individuals across the Americas. To complete its life cycle, T. cruzi parasitizes both vertebrate hosts and its vector, commonly known as the ‘kissing bug’. The parasite’s survival and proliferation strategies are driven by the diverse environments it encounters. Despite being described by Carlos Chagas in 1909, significant knowledge gaps persist regarding the parasite’s various life forms and adaptive capabilities in response to environmental cues. In this study, we employed Ultrastructure Expansion Microscopy to explore the intricate journey of T. cruzi within the host cell. Upon entry into the host cell, trypomastigotes undergo folding, resulting in intermediate forms characterized by a rounded cell body, anterior positioning of basal bodies, and a shortened flagellum. The repositioning of basal bodies and the kinetoplast and the shortening of the flagella mark the culmination of intracellular amastigogenesis. Furthermore, we analyzed intracellular trypomastigogenesis, identifying discrete intermediate forms, including leaf-shaped stages and epimastigote-like forms, which suggests a complex differentiation process. Notably, we did not observe any dividing intracellular epimastigotes, indicating that these may be non-replicative forms within the host cell. Our detailed examination of amastigote cell division revealed semi-closed nuclear mitosis, with mitotic spindle formation independent of basal bodies. This study provides new insights into the morphological and cytoskeletal changes during the intracellular stages of T. cruzi, providing a model for understanding the dynamics of intracellular amastigogenesis and trypomastigogenesis. Full article

Indazoles have previously been identified as molecules with antiprotozoal activity. In this study, we evaluate the in vitro activity of thirteen 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) against L. amazonensis, L. infantum, and L. mexicana. In vitro, cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for all compounds, and those showing adequate activity and selectivity were tested against intracellular amastigotes. Transmission and scanning electron microscopy were employed to study the effects of 3-alkoxy-1-benzyl-5-nitroindazole and 2-benzyl-5-nitroindazolin-3-one derivatives on promastigotes of L. amazonensis. Compounds NV6 and NV8 were active in the two life stages of the three species, with the latter showing the best indicators of activity and selectivity. 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) showed in vitro activity comparable to that of amphotericin B against the promastigote stage of Leishmania spp. Two compounds were also found to be active the amastigote stage. Electron microscopy studies confirmed the antileishmanial activity of the indazole derivatives studied and support future research on this family of compounds as antileishmanial agents. Full article

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action. Full article

Maintenance chemotherapy is a standard treatment in patients with non-progressive advance staged IV non-squamous non-small cell lung cancer after induction therapy. Here, we report the case of a 53-year-old man undergoing a maintenance monotherapy with pemetrexed who presented prolonged pancytopenia despite filgrastim injections. A bone marrow aspiration revealed a macrophage activation syndrome with Leishmania amastigotes. A Polymerase Chest Reaction testing confirmed the diagnosis of visceral leishmaniasis. Treatment with liposomal amphotericin B was started. Oncologists should bear in mind that visceral leishmaniasis in endemic areas can potentially induce severe and prolonged pancytopenia in immunosuppressed patients, during chemotherapy in particular. Full article

Chagas disease is one of the world’s neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC₅₀ values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC₅₀ determinations, and an assessment of structure–activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research. Full article

Cutaneous leishmaniasis (CL) is a common illness that causes skin lesions, principally ulcerations, on exposed regions of the body. Although neglected tropical diseases (NTDs) are typically found in tropical areas, they have recently become more common along Africa’s northern coast, particularly in Libya. The devastation of healthcare infrastructure during the 2011 war and the following conflicts, as well as governmental apathy, may be causal factors associated with this catastrophic event. The main objective of this study is to evaluate alternative diagnostic strategies for recognizing amastigotes of cutaneous leishmaniasis parasites at various stages using Convolutional Neural Networks (CNNs). The research is additionally aimed at testing different classification models employing a dataset of ultra-thin skin smear images of Leishmania parasite-infected people with cutaneous leishmaniasis. The pre-trained deep learning models including EfficientNetB0, DenseNet201, ResNet101, MobileNetv2, and Xception are used for the cutaneous leishmania parasite diagnosis task. To assess the models’ effectiveness, we employed a five-fold cross-validation approach to guarantee the consistency of the models’ outputs when applied to different portions of the full dataset. Following a thorough assessment and contrast of the various models, DenseNet-201 proved to be the most suitable choice. It attained a mean accuracy of 0.9914 along with outstanding results for sensitivity, specificity, positive predictive value, negative predictive value, F1-score, Matthew’s correlation coefficient, and Cohen’s Kappa coefficient. The DenseNet-201 model surpassed the other models based on a comprehensive evaluation of these key classification performance metrics. Full article

The use of artificial intelligence has shown good performance in the medical imaging area, in particular the deep learning methods based on convolutional neural networks for classification, detection, and/or segmentation tasks. The task addressed in this research work is the segmentation of amastigote nests from histological microphotographs in the study of Trypanosoma cruzi infection (Chagas disease) implementing a U-Net convolutional network architecture. For the nests’ segmentation, a U-Net architecture was trained on histological images of an acute-stage murine experimental model performing a 5-fold cross-validation, while the final tests were carried out with data unseen by the U-Net from three image groups of different experimental models. During the training stage, the obtained results showed an average accuracy of 98.19 ± 0.01, while in the case of the final tests, an average accuracy of 99.9 ± 0.1 was obtained for the control group, as well as 98.8 ± 0.9 and 99.1 ± 0.8 for two infected groups; in all cases, high sensitivity and specificity were observed in the results. We can conclude that the use of a U-Net architecture proves to be a relevant tool in supporting the diagnosis and analysis of histological images for the study of Chagas disease. Full article

Background: Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi. There is growing scientific interest in finding new and better therapeutic alternatives for this disease’s treatment. Methods: A total of 81 terpene compounds with potential trypanocidal activity were screened and found to have potential T. cruzi cysteine synthase (TcCS) inhibition using molecular docking, molecular dynamics, ADME and PAIN property analyses and in vitro susceptibility assays. Results: Molecular docking analyses revealed energy ranges from −10.5 to −4.9 kcal/mol in the 81 tested compounds, where pentacyclic triterpenes were the best. Six compounds were selected to assess the stability of the TcCS–ligand complexes, of which lupeol acetate (ACLUPE) and α-amyrin (AMIR) exhibited the highest stability during 200 ns of molecular dynamics analysis. Such stability was primarily due to their hydrophobic interactions with the amino acids located in the enzyme’s active site. In addition, ACLUPE and AMIR exhibited lipophilic characteristics, low intestinal absorption and no structural interferences or toxicity. Finally, selective index for ACLUPE was >5.94, with moderate potency in the trypomastigote stage (EC₅₀ = 15.82 ± 3.7 μg/mL). AMIR’s selective index was >9.36 and it was moderately potent in the amastigote stage (IC₅₀ = 9.08 ± 23.85 μg/mL). Conclusions: The present study proposes a rational approach for exploring lupeol acetate and α-amyrin terpene compounds to design new drugs candidates for Chagas disease. Full article

The second-line antileishmanial compound pentamidine is administered intramuscularly or, preferably, by intravenous infusion, with its use limited by severe adverse effects, including diabetes, severe hypoglycemia, myocarditis and renal toxicity. We sought to test the potential of phospholipid vesicles to improve the patient compliance and efficacy of this drug for the treatment of leishmaniasis by means of aerosol therapy. The targeting to macrophages of pentamidine-loaded liposomes coated with chondroitin sulfate or heparin increased about twofold (up to ca. 90%) relative to noncoated liposomes. The encapsulation of pentamidine in liposomes ameliorated its activity on the amastigote and promastigote forms of Leishmania infantum and Leishmania pifanoi, and it significantly reduced cytotoxicity on human umbilical endothelial cells, for which the concentration inhibiting 50% of cell viability was 144.2 ± 12.7 µM for pentamidine-containing heparin-coated liposomes vs. 59.3 ± 4.9 µM for free pentamidine. The deposition of liposome dispersions after nebulization was evaluated with the Next Generation Impactor, which mimics human airways. Approximately 53% of total initial pentamidine in solution reached the deeper stages of the impactor, with a median aerodynamic diameter of ~2.8 µm, supporting a partial deposition on the lung alveoli. Upon loading pentamidine in phospholipid vesicles, its deposition in the deeper stages significantly increased up to ~68%, and the median aerodynamic diameter decreased to a range between 1.4 and 1.8 µm, suggesting a better aptitude to reach the deeper lung airways in higher amounts. In all, nebulization of liposome-encapsulated pentamidine improved the bioavailability of this neglected drug by a patient-friendly delivery route amenable to self-administration, paving the way for the treatment of leishmaniasis and other infections where pentamidine is active. Full article

Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl₂(H3)₂ complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3β-ol, a well-known bioactive molecule functioning as a sterol Δ²⁴-sterol methyl transferase (24-SMT) inhibitor. The ZnCl₂(H3)₂ complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl₂(H3)₂ significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC₅₀ values found for H3 and ZnCl₂(H3)₂ were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl₂(H3)₂ complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl₂(H3)₂ (CC₅₀ = 5 μΜ, SI = 156) is more selective than H3 (CC₅₀ = 10 μΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3β-ol and cholesta-7,24-dien-3β-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl₂(H3)₂. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl₂(H3)₂. Full article