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The problems with current strategies to control canine visceral Leishmaniasis (CVL), which include the euthanasia ofinfected animals, and also the toxicity of the drugs currently used in human treatments for CVL, add urgency to the search for newtherapeutic agents. This study aimed to evaluate the activity against Leishmania (L.) infantum of 12 amides that are chemicallyinspired by gibbilimbol B, a bioactive natural product that was initially obtained from Piper malacophyllum. Three of thesecompounds N-(2-ethylhexyl)-4-chlorobenzamide (9), N-(2-ethylhexyl)-4-nitrobenzamide (10), and N-(2-ethylhexyl)-4-(tert-butyl)benzamide (12) demonstrated activity against the intracellular amastigotes without toxicity to mammalian host cells (CC50> 200 μM); compounds 9, 10, and 12 resulted in EC50 values of 12.7, 12.2, and 5.1 μM, respectively. In silico drug-likeness studiespredicted that these compounds would show high levels of gastrointestinal absorption, would be able to penetrate the blood-brainbarrier, would show moderate solubility, and would not show unwanted molecular interactions. Due to their promisingpharmacological profiles, compounds 9 and 10 were selected for mechanism of action studies (MoA). The MoA studies in L. (L.)infantum revealed that neither of the compounds affected the permeabilization of the plasma membrane. Nevertheless, compound 9induced strong alkalinization of acidocalcisomes, which resulted in a significant and rapid increase in intracellular Ca2+ levels, therebycausing the depolarization of the mitochondrial membrane potential and a reduction in the levels of reactive oxygen species (ROS).In contrast, compound 10 induced a gradual increase in intracellular Ca2+ levels and a similarly gradual reduction in ROS levels, butit caused neither acidocalcisome alkalinization nor mitochondrial membrane potential depolarization. Finally, the MALDI-TOF/MSassessment of protein alterations in L. (L.) infantum treated separately with compounds 9 and 10 revealed changes in mass spectralprofiles from both treatments. These results highlight the anti-L. (L.) infantum potential of these amides especially for compounds9 and 10 and they suggest that these compounds could be promising candidates for future in vivo studies in VL-models.
RESUMEN
As part of our continuous research for the discovery of bioactive compounds against Trypanosoma cruzi and Leishmania infantum, the alkaloid (6aS)-dicentrine (1) was oxidized to afford (6aS,6S)- (2) and (6aS,6R)- (3) dicentrine-N-oxides. Evaluation of the cytotoxicity against NCTC cells indicated that 2 and 3 are non-toxic (CC50>200 μM) whereas 1 demonstrated CC50 of 52.0 μM. Concerning T. cruzi activity against amastigotes, derivatives 2 and 3 exhibited EC50 values of 9.9 μM (SI>20.2) and 27.5 μM (SI>7.3), respectively, but 1 is inactive (EC50>100 μM). Otherwise, when tested against L. infantum amastigotes, 1 and 3 exhibited EC50 values of 10.3 μM (SI=5.0) and 12.7 μM (SI>15.7), respectively, being 2 inactive (EC50>100 μM). Comparing the effects of positive controls benznidazol (EC50=6.5 μM and SI>30.7) and miltefosine (EC50=10.2 μM and SI=15.2), it was observed a selective antiparasitic activity to diastereomers 2 and 3 against T. cruzi and L. infantum. Considering stereochemical aspects, it was suggested that the configuration of the new stereocenter formed after oxidation of 1 played an important role in the bioactivity against amastigotes of both tested parasites.
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Neglected Tropical Diseases are a significant concern as theyencompass various infections caused by pathogens prevalent intropical regions. The limited and often highly toxic treatmentoptions for these diseases necessitate the exploration of newtherapeutic candidates. In the present study, the lignanmethylpiperitol was isolated after several chromatographicsteps from Persea fulva L. E. Koop (Lauraceae) and its leishmani-cidal and trypanocidal activities were evaluated using in vitroand in silico approaches. The chemical structure of methylpiper-itol was defined by NMR and MS spectral data analysis. Theantiprotozoal activity of methylpiperitol was determined in vitroand indicated potency against trypomastigote forms of Trypa-nosoma cruzi (EC50 of 4.5 � 1.1 mM) and amastigote forms ofLeishmania infantum (EC50 of 4.1 � 0.5 mM), with no mammaliancytotoxicity against NCTC cells (CC50 > 200 mM). Moleculardocking studies were conducted using six T. cruzi and fourLeishmania. The results indicate that for the molecular targethypoxanthine phosphoribosyl transferase in T. cruzi and pite-ridine reductase 1 of L. infatum, the methylpiperitol obtainedbetter results than the crystallographic ligand. Therefore, thelignan methylpiperitol, isolated from P. fulva holds potential forthe development of new prototypes for the treatment ofNeglected Tropical Diseases, especially leishmaniasis.
RESUMEN
Chagas disease is a Neglected Tropical Disease with limited and ineffective therapy. In a search for new anti-trypanosomal compounds, we investigated the potential of the metabolites from the bacteria living in the corals and sediments of the southeastern Brazilian coast. Three corals, Tubastraea coccinea, Mussismilia hispida, Madracis decactis, and sediments yielded 11 bacterial strains that were fully identified by MALDI-ToF/MS or gene sequencing, resulting in six genera—Vibrio, Shewanella, Mesoflavibacter, Halomonas, Bacillus, and Alteromonas. To conduct this study, EtOAc extracts were prepared and tested against Trypanosoma cruzi. The crude extracts showed IC50 values ranging from 15 to 51 μg/mL against the trypomastigotes. The bacterium Mesoflavibacter zeaxanthinifaciens was selected for fractionation, resulting in an active fraction (FII) with IC50 values of 17.7 μg/mL and 23.8 μg/mL against the trypomastigotes and amastigotes, respectively, with neither mammalian cytotoxicity nor hemolytic activity. Using an NMR and ESI-HRMS analysis, the FII revealed the presence of unsaturated iso-type fatty acids. Its lethal action was investigated, leading to a protein spectral profile of the parasite altered after treatment. The FII also induced a rapid permeabilization of the plasma membrane of the parasite, leading to cell death. These findings demonstrate that these unsaturated iso-type fatty acids are possible new hits against T. cruzi.
RESUMEN
This study aimed to assess the antiprotozoal efficacy of dicentrine, an aporphine alkaloid isolated from Ocotea puberula, against amastigote forms of Leishmania (L.) infantum. Our findings reveal that dicentrine demonstrated a notable EC50 value of 10.3 μM, comparable to the positive control miltefosine (EC50 of 10.4 μM), while maintaining moderate toxicity to macrophages (CC50 of 51.9 μM). Utilizing an in silico methodology, dicentrine exhibited commendable adherence to various parameters, encompassing lipophilicity, water solubility, molecule size, polarity, and flexibility. Subsequently, we conducted additional investigations to unravel the mechanism of action, employing Langmuir monolayers as models for protozoan cell membranes. Tensiometry analyses unveiled that dicentrine disrupts the thermodynamic and mechanical properties of the monolayer by expanding it to higher areas and increasing the fluidity of the film. The molecular disorder was further corroborated through dilatational rheology and infrared spectroscopy. These results contribute insights into the role of dicentrine as a potential antiprotozoal drug in its interactions with cellular membranes. Beyond elucidating the mechanism of action at the plasma membrane's external surface, our study sheds light on drug-lipid interface interactions, offering implications for drug delivery and other pharmaceutical applications.
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The hexane extract from twigs of Piper truncatum Vell (Piperaceae) displayed activity against Trypanosoma cruzi and was subjected to chromatographic steps to afford six dibenzylbutyrolactolic lignans, being four knowns: cubebin (1), (−)-9α-O-methylcubebin (2), (+)-9β-O-methylcubebinin (3) and 3,4-dimethoxy-3,4-demethylenedioxycubebin (4) as well as two new, named truncatin A (5) and B (6). Initially, in vitro activity against trypomastigotes was evaluated and compounds 1, 4 and 6 exhibited EC50 values of 41.6, 21.0 and 39.6 μM, respectively. However, when tested against amastigotes, the relevant clinical form in the chronic phase of Chagas disease, compounds 1–6 displayed activities with EC50 values ranging from 1.6 to 13.7 μM. In addition, the mammalian cytotoxicity of compounds 1–6 was evaluated against murine fibroblasts (NCTC). Compounds 2, 3 and 4 exhibited reduced toxicity against NCTC cells (CC50>200 μM), resulting in SI values of>21.9,>14.5 and>121.9, respectively. Compound 4 showed the highest potency with an SI value twice superior to that determined by the standard drug benznidazole (SI>54.6) against the intracellular amastigotes. These data suggest that lignan 4 can be considered a possible scaffold for designing a new drug candidate for Chagas disease.
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In the present work, the hexane extract of aerial parts of Baccharis quitensis Kunth. was subjected to chromatographic fractionation to afford two alkyl phenylpropanoids: n-docosyl (E)-p-coumarate (1) and n-tetracosyl (E)-p-coumarate (2) as well as five diterpenes: ent-kaurenoic acid (3), grandifloric acid (4), 15β-senecioyl-oxy-ent-kaur-16-en-19-oic acid (5), and 15-oxo-ent-kaurenoic acid (6). Using an ex-vivo assay with macrophages infected with Trypanosoma cruzi, compounds 1 and 2 demonstrated high potency against intracellular amastigotes, with EC50 values of 7.5 and 6.9 µM, respectively. Compound 6 revealed a moderate potency against T. cruzi, with an EC50 of 25.6 µM, and compounds 3–5 showed no effectiveness. Additionally, compounds 1–6 compounds presented no toxicity for mammalian cells to the highest tested concentration of 200 µM. Based on potency and the selectivity indexes of 1, 2 and 6, these compounds could be future candidates for optimisation studies for the design of prototypes against Chagas disease.
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Visceral leishmaniasis and Chagas disease are neglected tropical diseases (NTDs) that severely impact the developing world. With current therapies suffering from poor efficacy and safety profiles as well as emerging resistance, new drug leads are direly needed. In this work, 26 alkaloids (9 natural and 17 synthetic) belonging to the benzyltetrahydroisoquinoline (BI) family were evaluated against both the pro/trypomastigote and amastigote forms of the parasites Leishmania infantum and Trypanosoma cruzi, the causative agents of these diseases. These alkaloids were synthesized via an efficient and modular enantioselective approach based on Bischler-Napieralski cyclization/Noyori asymmetric transfer hydrogenation to build the tetrahydroisoquinoline core. The bis-benzyltetrahydroisoquinoline (BBI) alkaloids were prepared using an Ullmann coupling of two BI units to form the biaryl ether linkage, which enabled a comprehensive survey of the influence of BI stereochemistry on bioactivity. Preliminary studies into the mechanism of action against Leishmania mexicana demonstrate that these compounds interfere with the cell cycle, potentially through inhibition of kinetoplast division, which may offer opportunities to identify a new target/mechanism of action. Three of the synthesized alkaloids showed promising druglike potential, meeting the Drugs for Neglected Disease initiative (DNDi) criteria for a hit against Chagas disease.
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Background Chagas disease and leishmaniasis affect a significant portion of the Latin American population and still lack efficient treatments. In this context, natural products emerge as promising compounds for developing more effective therapies, aiming to mitigate side effects and drug resistance. Notably, species from the Amaryllidaceae family emerge as potential reservoirs of antiparasitic agents due to the presence of diverse biologically active alkaloids. Purpose To assess the anti-Trypanosoma cruzi and anti-Leishmania infantum activity of five isolated alkaloids from Hippeastrum aulicum Herb. (Amaryllidaceae) against different life stages of the parasites using in silico and in vitro assays. Furthermore, molecular docking was employed to evaluate the interaction of the most active alkaloids. Methods Five natural isoquinoline alkaloids isolated in suitable quantities for in vitro testing underwent preliminary in silico analysis to predict their potential efficacy against Trypanosoma cruzi (amastigote and trypomastigote forms) and Leishmania infantum (amastigote and promastigote forms). The in vitro antiparasitic activity and mammalian cytotoxicity were investigated with a subsequent comparison of both analysis (in silico and in vitro) findings. Additionally, this study employed the molecular docking technique, utilizing cruzain (T. cruzi) and sterol 14α-demethylase (CYP51, L. infantum) as crucial biological targets for parasite survival, specifically focusing on compounds that exhibited promising activities against both parasites. Results Through computational techniques, it was identified that the alkaloids haemanthamine (1) and lycorine (8) were the most active against T. cruzi (amastigote and trypomastigote) and L. infantum (amastigote and promastigote), while also revealing unprecedented activity of alkaloid 7‑methoxy-O-methyllycorenine (6). The in vitro analysis confirmed the in silico tests, in which compound 1 presented the best activities against the promastigote and amastigote forms of L. infantum with half-maximal inhibitory concentration (IC50) 0.6 µM and 1.78 µM, respectively. Compound 8 exhibited significant activity against the amastigote form of T. cruzi (IC50 7.70 µM), and compound 6 demonstrated activity against the trypomastigote forms of T. cruzi and amastigote of L. infantum, with IC50 values of 89.55 and 86.12 µM, respectively. Molecular docking analyses indicated that alkaloids 1 and 8 exhibited superior interaction energies compared to the inhibitors. Conclusion The hitherto unreported potential of compound 6 against T. cruzi trypomastigotes and L. infantum amastigotes is now brought to the forefront. Furthermore, the acquired dataset signifies that the isolated alkaloids 1 and 8 from H. aulicum might serve as prototypes for subsequent structural refinements aimed at the exploration of novel leads against both T. cruzi and L. infantum parasites.
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Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene — sphenophyllol (1) — as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O caffeoylquinic acids (6–8). Compounds 1–8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC50 values of 20.1 µM and 2.9 µM, respectively. The mixture of chlorogenic acids 6–8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC50 values of 24.9, 12.8, and 2.7 µM, respectively. Nonetheless, tested compounds 1–8 displayed no cytotoxicity against mammalian cells (CC50 > 200 µM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases.
RESUMEN
American Trypanosomiasis, also known as Chagas disease, is caused by the protozoan Trypanosoma cruzi and exhibits limited options for treatment. Natural products offer various structurally complex metabolites with biological activities, including those with anti-T. cruzi potential. The discovery and development of prototypes based on natural products frequently display multiple phases that could be facilitated by machine learning techniques to provide a fast and efficient method for selecting new hit candidates. Using Random Forest and k-Nearest Neighbors, two models were constructed to predict the biological activity of natural products from plants against intracellular amastigotes of T. cruzi. The diterpenoid andrographolide was identified from a virtual screening as a promising hit compound. Hereafter, it was isolated from Cymbopogon schoenanthus and chemically characterized by spectral data analysis. Andrographolide was evaluated against trypomastigote and amastigote forms of T. cruzi, showing IC50 values of 29.4 and 2.9 μM, respectively, while the standard drug benznidazole displayed IC50 values of 17.7 and 5.0 μM, respectively. Additionally, the isolated compound exhibited a reduced cytotoxicity (CC50 = 92.8 μM) against mammalian cells and afforded a selectivity index (SI) of 32, similar to that of benznidazole (SI = 39). From the in silico analyses, we can conclude that andrographolide fulfills many requirements implemented by DNDi to be a hit compound. Therefore, this work successfully obtained machine learning models capable of predicting the activity of compounds against intracellular forms of T. cruzi.
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Melanin is a substance that plays important roles in several organisms. Its function as an antioxidant and metal-complexing agent makes tyrosinase, the key enzyme that controls melanogenesis, an interesting target for designing inhibitors. In this article, we report a set of piperazine/piperidine amides of benzoic and cinnamic acid derivatives as tyrosinase inhibitors with improved potency and drug-likeness. The most potent compound 5b showed a pIC50 of 4.99 in the monophenolase assay, and only compound 3a showed reasonable potency in the diphenolase assay (pIC50, 4.18). These activities are not correlated to antiradical activity, suggesting that the activity is dependent on competition with the substrates. Molecular docking studies indicated that the benzyl substituent of 5b and other analogues perform important interactions in the enzyme that may explain the higher potency of these compounds. Moreover, the compounds present adequate lipophilicity and skin permeability and no relevant cytotoxicity (CC50 > 200 μM) to mammalian cells.