Andrographolide: A Diterpenoid from Cymbopogon schoenanthus Identified as a New Hit Compound against Trypanosoma cruzi Using Machine Learning and Experimental Approaches.

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.

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis.

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.

Repurposing topical triclosan for cutaneous leishmaniasis: Preclinical efficacy in a murine Leishmania (L.) amazonensis model.

Leishmaniasis remains an important neglected tropical infection caused by the protozoan Leishmania and affects 12 million people in 98 countries. The treatment is limited with severe adverse effects. In the search for new therapies, the drug repositioning and combination therapy have been successfully applied to neglected diseases. The aim of the present study was to evaluate the in vitro and in vivo anti-Leishmania (Leishmania) amazonensis potential of triclosan, an approved topical antimicrobial agent used for surgical procedures. in vitro phenotypic studies of drug-treated parasites were performed to evaluate the lethal action of triclosan, accompanied by an isobolographic ex-vivo analysis with the association of triclosan and miltefosine. The results showed that triclosan has activity against L. (L.) amazonensis intracellular amastigotes, with a 50% inhibitory concentration of 16 µM. By using fluorescent probes and transmission electron microscopy, a pore-forming activity of triclosan toward the parasite plasma membrane was demonstrated, leading to depolarization of the mitochondrial membrane potential and reduction of the reactive oxygen species levels in the extracellular promastigotes. The in vitro interaction between triclosan and miltefosine in the combination therapy assay was classified as additive against intracellular amastigotes. Leishmania-infected mice were treated with topical triclosan (1% base cream for 14 consecutive days), and showed 89% reduction in the parasite burden. The obtained results contribute to the investigation of new alternatives for the treatment of cutaneous leishmaniasis and suggest that the coadministration of triclosan and miltefosine should be investigated in animal models.

Antitrypanosomal activity of epi-polygodial from Drimys brasiliensis and its effects in cellular membrane models at the air-water interface.

Epi-polygodial, a drimane sesquiterpene was isolated from Drimys brasiliensis (Winteraceae). This compound demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (IC50 = 5.01 µM) with a selectivity index higher than 40. These results were correlated with the effects observed when this compound was incorporated in cellular membrane models of protozoans, represented by Langmuir monolayers of dipalmitoylphosphoethanolamine (DPPE). Surface pressure-area isotherms showed that epi-polygodial expands DPPE monolayers at higher areas and condenses them at lower areas, which was attributed to the preferential interaction with the polar heads of the lipid. This mechanism of action could be corroborated with Polarization-Modulation Reflection-Absorption Spectroscopy and Brewster Angle Microscopy. These results pointed to the fact that the interaction of epi-polygodial with DPPE monolayers at the air-water interface affects the physical chemical properties of the mixed film, which may be important to comprehend the interaction of this drug with cellular membranes at the molecular level.

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model.

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the ζ-potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.

Activity of imidazole compounds on Leishmania (L.) infantum chagasi: reactive oxygen species induced by econazole.

Drug repositioning has been considered a promising approach to discover novel treatments against neglected diseases. Among the major protozoan diseases, leishmaniasis remains a public health threat with few therapeutic alternatives, affecting 12 million people in 98 countries. In this study, we report the in vitro antileishmanial activity of the imidazole drugs clotrimazole, and for the first time in literature, econazole and bifonazole and their potential action to affect the regulation of reactive oxygen species (ROS) of the parasites. The lethal action of the imidazoles was investigated using spectrofluorimetric techniques to detect ROS content, plasma membrane permeability, and mitochondrial membrane potential. The imidazoles showed activity against L. (L.) infantum chagasi promastigotes with IC50 values in a range of 2-8 µM; econazole was also effective against Leishmania intracellular amastigotes, with an IC50 value of 11 µM, a similar in vitro effectiveness to miltefosine. Leishmania promastigotes rapidly up-regulated the ROS release after incubation with the imidazoles, but econazole showed a marked increase in ROS content of approximately 1,900 % higher than untreated parasites. When using SYTOX(®) Green as a fluorescent probe, the imidazoles demonstrated considerable interference in plasma membrane permeability at the early time of incubation; econazole resulted in the higher influx of SYTOX(®) Green at 60 min. Despite cellular alterations, no depolarization could be observed to the mitochondrial membrane potential of Leishmania until 60 min. The lethal action of econazole involved strong permeabilization of plasma membrane of promastigotes, with an overloaded ROS content that contributed to the death of parasites. Affecting the ROS regulation of Leishmania via small molecules would be an interesting strategy for new drugs.

Insulin-like growth factor-I induces arginase activity in Leishmania amazonensis amastigote-infected macrophages through a cytokine-independent mechanism.

Leishmania (Leishmania) amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF)-I on interactions between L. (L.) amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS) exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-ß antibodies. Our results suggest that in L. (L.) amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

Steam distillation, supercritical fluid extraction, and anti-Trypanosoma cruzi activity of compounds from pink pepper (Schinus terebinthifolius Raddi).

Anti-Trypanosoma cruzi activity of compounds from fruits of Schinus terebinthifolius Raddi (pink pepper) were evaluated, using sustainable techniques such as steam distillation (SD) and supercritical fluid extraction (SFE). SD was optimised using a design of experiment and SFE was carried out using supercritical CO2 solvent (300 bar and 60 °C). Results of the anti-T. cruzi activity showed that the essential oil presented high activity (IC50 = 4.5 ± 0.3 µg/mL), whereas the supercritical extract had a moderate effect (IC50 = 19.7 ± 2.9 µg/mL). The differences in the anti-T. cruzi activity can be attributed to the extraction of non-volatile compounds in the SFE, such as moronic and (Z)-masticadienoic acids. In contrast, SD extracted only volatile compounds such as monoterpenes and sesquiterpenes. Therefore, these results suggest that the volatile compounds from pink pepper are involved with the anti-T. cruzi activity.

Multitarget anti-parasitic activities of isoquinoline alkaloids isolated from Hippeastrum aulicum (Amaryllidaceae).

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 7­methoxy-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.

Saturated Iso-Type Fatty Acids from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens with Anti-Trypanosomal Potential.

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.

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi.

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25-0.68 µM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 µM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50=34 µM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX(®) Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.

Identification of inhibitors as drug candidates against Chagas disease.

Chagas disease, after more than a century after its discovery, is still a major public health problem. It is estimated that approximately 10 million people worldwide are infected with T. cruzi. However, the situation is more critical in Latin America and other regions where the disease is endemic. The largest number of cases occurs in Brazil, Argentina, and Mexico as more than 100 million people in these regions are located in areas with a high risk of contamination by the vector. The need for new therapeutic alternatives is urgent, as the available drugs have severe limitations such as low efficacy and high toxicity. From this scenario, in this work, we employed the virtual screening technique using cruzain and BDF2 as key biological targets for the survival of the parasite. Our objective was to identify potential inhibitors of T. cruzi trypomastigotes, which could be considered drug candidates against Chagas disease. For this, we employed different in silico methodologies and the obtained results were corroborated using in vitro biological assays. For the VS studies, a database containing synthetic compounds was simulated at the binding site of cruzain and BDF2. In addition, pharmacophoric models were constructed in the initial phases of VS, as well as other advanced analyses (molecular dynamics simulations, calculations of binding free energy, and ADME prediction) were carried out and the results allowed the selection of potential inhibitors of T. cruzi. Based on the obtained data, 32 different compounds commercially available were subjected to biological tests against the trypomastigote form of T. cruzi. As result, 11 of those compounds displayed significant activity against T. cruzi and can be considered potential candidates for the treatment of Chagas disease.

The Potential of Secondary Metabolites from Plants as Drugs or Leads against Trypanosoma cruzi-An Update from 2012 to 2021.

BACKGROUND: Chagas disease (American Trypanosomiasis) is classified by the World Health Organization (WHO) as one of the seventeen neglected tropical diseases (NTD), affecting, mainly, several regions of Latin America. INTRODUCTION: However, immigration has expanded the range of this disease to other continents. Thousands of patients with Chagas disease die annually, yet no new therapeutics for Chagas disease have been approved, with only nifurtimox and benznidazole available. Treatment with these drugs presents several challenges, including protozoan resistance, toxicity, and low efficacy. Natural products, including the secondary metabolites found in plants, offer a myriad of complex structures that can be sourced directly or optimized for drug discovery. METHODS: Therefore, this review aims to assess the literature from the last 10 years (2012-2021) and present the anti-T. cruzi compounds isolated from plants in this period, as well as briefly discuss computational approaches and challenges in natural product drug discovery. Using this approach, more than 350 different metabolites were divided based on their biosynthetic pathway alkaloids, terpenoids, flavonoids, polyketides, and phenylpropanoids which displayed activity against different forms of this parasite epimastigote, trypomastigote and more important, the intracellular form, amastigote. CONCLUSION: In this aspect, there are several compounds with high potential which could be considered as a scaffold for the development of new drugs for the treatment of Chagas disease-for this, more advanced studies must be performed including pharmacokinetics (PK) and pharmacodynamics (PD) analysis as well as conduction of in vivo assays, these being important limitations in the discovery of new anti-T. cruzi compounds.

Effect of partial O-methylation in dehydrodieugenol on its antitrypanosomal activity - correlation with the toxicity using cell membrane models.

Biseugenol (1), a neolignan with antiprotozoal activity against Trypanosoma cruzi, was partially methylated, and the compound obtained - methyl biseugenol (2) - had its activity evaluated against the extracellular (trypomastigotes) and intracellular (amastigotes) forms of T. cruzi. It was observed that both compounds 1 and 2 exhibited similar effects against trypomastigotes (IC50 of 11.7 and 16.2 µM, respectively), whereas compound 2 displayed higher activity against amastigotes (IC50 = 8.2 µM) in comparison with biseugenol (IC50 = 15.4 µM). Additionally, reduced toxicity against NCTC cells for compound 2 was observed (CC50 > 200 µM), differently from compound 1 with CC50 = 58.0 µM. Aiming to understand better the molecular mechanism of the biological action of compound 2, the prodrug was incorporated into cellular membrane models constituted of Langmuir monolayers of the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG). The lipid-drug interaction was inferred through tensiometry, surface potential, infrared spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The prodrug expanded DPPC and DPPG monolayers and condensed DPPE ones, as well as presented characteristic behaviors regarding the chemical structure of the lipid considering expansion-compression curves, surface potential-area isotherms, and stability of previously compressed monolayers to relevant-biological surface pressures. PM-IRRAS indicated a molecular disorder for DPPC and DPPS alkyl chains in the presence of the drug. BAM revealed the presence of domains in the DPPG and DPPE monolayers, which was probably induced by the prodrug. These data suggest, in general, that the lipid composition modulates the interaction of compound 2, whose results are expected to correlate to its trypanocidal activity, which involves the plasma membrane of T. cruzi as the primary target, i.e., the first barrier that the compound should encounter to interact with the microorganism.

Evaluation of antileishmanial potential of the antidepressant escitalopram in Leishmania infantum.

Neglected tropical diseases (NTDs) such as visceral leishmaniasis (VL) present a limited and toxic therapeutic arsenal, and drug repositioning represents a safe and cost-effective approach. In this work, we investigated the antileishmanial potential and the mechanism of lethal action of the antidepressant escitalopram. The efficacy of escitalopram was determined ex-vivo using the intracellular Leishmania (L.) infantum amastigote model and the mammalian cytotoxicity was determined by the colorimetric MTT assay. The cellular and molecular alterations induced by the drug were investigated using spectrofluorimetry, a luminescence assay and flow cytometry. Our data revealed that escitalopram was active and selective against L. infantum parasites, with an IC50 value of 25 µM and a 50% cytotoxic concentration (CC50) of 184 µM. By using the fluorescent probes SYTOX® Green and DiSBAC2(3), the drug showed no alterations in the plasma membrane permeability nor in the electric potential of the membrane (∆ψp); however, after a short-time incubation, the drug caused a dose-dependent up-regulation of the calcium levels, leading to the depolarization of the mitochondrial membrane potential (∆ψm) and a reduction of the ATP levels. No up-regulation of reactive oxygen (ROS) was observed. In the cell cycle analysis, escitalopram induced a dose-dependent increase of the parasites at the sub G0/G1 stage, representing fragmented DNA. Escitalopram presented a selective antileishmanial activity, with disruption of single mitochondrion and interference in the cell cycle. Approved drugs such as escitalopram may represent a promising approach for NTDs and can be considered in future animal efficacy studies.

A new reduced chalcone-derivative affects the membrane permeability and electric potential of multidrug-resistant Enterococcusfaecalis.

The emergence and spread of multidrug-resistant (MDR) enterococci and other Gram-positive bacteria represents a severe problem due to the lack of effective therapeutic alternatives. Natural products have long been an important source of new antibacterial scaffolds and can play a key role in the current antibiotic crisis. Enterococci are predominantly non-pathogenic gastrointestinal commensal bacteria, but among them, Enterococcus faecalis and Enterococcus faecium represent the species that account for most clinically relevant infections. The emergence of MDR enterococci has reduced the available antibiotic treatment options and highlights the need to develop new antimicrobial compounds. In the search for new hit compounds against MDR Enterococcus spp., natural-derived compounds represent inspiring scaffolds for drug design studies. In this work, the antimicrobial activity of a fully synthetic chalcone derivative (r4MB) was determined on a clinical panel of 34 MDR Gram-positive bacteria, mostly constituted by E. faecalis and E. faecium, along with Staphylococcus spp., amongst others. Compound r4MB showed activity against 100% of the tested strains, with the minimum inhibitory concentration (MIC) in the range of 5-20 µM. The lethal action of the compound was evaluated using different fluorescent-based assays. The compound showed a time-dependent permeabilisation of the membrane of a vancomycin-resistant E. faecalis, detected by the fluorescent probe SYTOX Green, and digital fluorescent microscopy corroborated the spectrofluorimetric analysis within 6 min of incubation. Flow cytometry analysis of the membrane electric potential demonstrated a significant depolarization, confirming the target of the compound towards the bacterial membrane. No cytotoxic haemolysis was observed with mammalian erythrocytes, and a 99% cytotoxic concentration of 118 µM on NCTC cells demonstrated a promising antimicrobial selectivity. In silico studies using SwissADME and ADMETLabs servers suggest that compound r4MB displayed adequate ADME properties, with no alerts for pan-assay interference compounds (PAINS). Future hit-to-lead optimization of this chalcone derivative can contribute to developing a more potent derivative against infections caused by MDR enterococci.

Investigation into in vitro anti-leishmanial combinations of calcium channel blockers and current anti-leishmanial drugs.

The need for drug combinations to treat visceral leishmaniasis (VL) arose because of resistance to antimonials, the toxicity of current treatments and the length of the course of therapy. Calcium channel blockers (CCBs) have shown anti-leishmanial activity; therefore their use in combination with standard drugs could provide new alternatives for the treatment of VL. In this work, in vitro isobolograms of Leishmania (Leishmania) chagasi using promastigotes or intracellular amastigotes were utilised to identify the interactions between five CCBs and the standard drugs pentamidine, amphotericin B and glucantime. The drug interactions were assessed with a fixed ratio isobologram method and the fractional inhibitory concentrations (FICs), sum of FICs (ΣFICs) and the overall mean ΣFIC were calculated for each combination. Graphical isobologram analysis showed that the combination of nimodipine and glucantime was the most promising in amastigotes with an overall mean ΣFIC value of 0.79. Interactions between CCBs and the anti-leishmanial drugs were classified as indifferent according to the overall mean ΣFIC and the isobologram graphic analysis.

Bioenergetics impairment of Trypanosoma cruzi by the antihypertensive manidipine: A drug repurposing strategy.

Considering the lack of effective and safe therapy for the treatment of Chagas disease, the antihypertensive drug manidipine (MDP) was in vitro evaluated against Trypanosoma cruzi. The bioenergetics of trypomastigotes was studied in the presence of the drug using fluorimetric and luminescent assays. Manidipine showed a potent antiparasitic activity, with IC50 values of 0.1 µM (intracellular amastigotes) and 3 µM (trypomastigotes), resulting in a promising selectivity index against the amastigotes (>1459). Using fluorimetric analysis, the drug showed depolarisation of the electric potential of the plasma membrane with no alteration of the permeability. A decrease in ATP levels suggested a bioenergetic alteration of the mitochondria, which was confirmed by the depolarisation of the mitochondrial membrane potential and a slight increase of the ROS levels. This is the first study to show the promising in vitro effectiveness of the antihypertensive MDP against T. cruzi, which may represent a candidate for future investigations in animal models.

Antitrypanosomal activity of a diterpene and lignans isolated from Aristolochia cymbifera.

Bioguided fractionation of extract from the leaves of Aristolochia cymbifera led to the isolation of the furofuran lignans fargesin, epieudesmin, and sesamin; the dibenzylbutyrolactone lignans hinokinin and kusunokinin; and an ENT-labdane diterpene named copalic acid. Our data demonstrated that copalic acid and kusunokinin were the most active compounds against trypomastigotes of Trypanosoma cruzi. Additionally, copalic acid demonstrated the highest parasite selectivity as a result of low toxicity to mammalian cells, despite a considerable hemolytic activity at higher concentrations. Among the isolated compounds, kusunokinin could be considered the most promising candidate, as it displayed significant activity against intracellular amastigotes (IC(50) = 17 µM) and trypomastigotes (IC(50) = 51 µM) without hemolytic activity. Fargesin, hinokinin, epieudesmin, and sesamin were also effective against trypomastigotes, but these compounds were highly toxic to mammalian cells and no parasite selectivity could be identified. The need for novel drugs for American trypanosomiasis is evident, and these secondary metabolites from A. cymbifera represent a useful tool for drug design.

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study.

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC(50) values ranging between 0.47 and 0.73 microg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.