Selective Activity against Amastigote Forms of Trypanosoma cruzi and Leishmania infantum of Diasteriomeric Dicentrine N-oxides.

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 > 20.7) 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 = 15.0), 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.

Piperazine amides with desirable solubility, physicochemical and drug-like properties: Synthesis and evaluation of the anti-Trypanosoma cruzi activity.

The absence of effective chronic treatment, expansion to non-endemic countries and the significant burden in public health have stimulated the search for novel therapeutic options to treat Chagas disease, a protozoan disease caused by Trypanosoma cruzi. Despite current efforts, no new drug candidates were approved in clinical trials in the past five decades. Considering this, our group has focused on the expansion of a series (LINS03) with low micromolar activity against amastigotes, considering the optimization of pharmacokinetic properties through increasing drug-likeness and solubility. In this work, we report a new set of 13 compounds with modifications in both the arylpiperazine and the aromatic region linked by an amide group. Five analogues showed activity against intracellular amastigotes (IC50 17.8 to 35.9 µM) and no relevant cytotoxicity to mammalian cells (CC50 > 200 µM). Principal component analysis (PCA) was performed to identify structural features associated to improved activity. The data revealed that polarity, hydrogen bonding ability and flexibility were key properties that influenced the antiparasitic activity. In silico drug-likeness assessments indicated that compounds with the 4-methoxycinammyl (especially compound 2b) had the most prominent balance between properties and activity in the series, as confirmed by SAR analysis.

Mitochondrial Imbalance of Trypanosoma cruzi Induced by the Marine Alkaloid 6-Bromo-2'-de-N-Methylaplysinopsin.

Chagas disease, caused by Trypanosoma cruzi, affects seven million people worldwide and lacks effective treatments. Using bioactivity-guided fractionation, NMR, and electrospray ionization-high resolution mass spectrometry (ESI-HRMS) spectral analysis, the indole alkaloid 6-bromo-2'-de-N-methylaplysinopsin (BMA) was isolated and chemically characterized from the marine coral Tubastraea tagusensis. BMA was tested against trypomastigotes and intracellular amastigotes of T. cruzi, resulting in IC50 values of 62 and 5.7 µM, respectively, with no mammalian cytotoxicity. The mechanism of action studies showed that BMA induced no alterations in the plasma membrane permeability but caused depolarization of the mitochondrial membrane potential, reducing ATP levels. Intracellular calcium levels were also reduced after the treatment, which was associated with pH alteration of acidocalcisomes. Using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/MS analysis, alterations of mass spectral signals were observed after treatment with BMA, suggesting a different mechanism from benznidazole. In silico pharmacokinetic-pharmacodynamic (PKPD) parameters suggested a drug-likeness property, supporting the promising usefulness of this compound as a new hit for optimizations.

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.

Antileishmanial Effects of Acetylene Acetogenins from Seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae) and Semisynthetic Derivatives.

As part of our continuous studies involving the prospection of natural products from Brazilian flora aiming at the discovery of prototypes for the development of new antiparasitic drugs, the present study describes the isolation of two natural acetylene acetogenins, (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-yn-19'-enyl)butanolide (1) and (2S,3R,4R)-3-hydroxy-4-methyl-2-(n-eicos-11'-ynyl)butanolide (2), from the seeds of Porcelia macrocarpa (Warm.) R.E. Fries (Annonaceae). Using an ex-vivo assay, compound 1 showed an IC50 value of 29.9 µM against the intracellular amastigote forms of Leishmania (L.) infantum, whereas compound 2 was inactive. These results suggested that the terminal double bond plays an important role in the activity. This effect was also observed for the semisynthetic acetylated (1a and 2a) and eliminated (1b and 2b) derivatives, since only compounds containing a double bond at C-19 displayed activity, resulting in IC50 values of 43.3 µM (1a) and 23.1 µM (1b). In order to evaluate the effect of the triple bond in the antileishmanial potential, the mixture of compounds 1 + 2 was subjected to catalytic hydrogenation to afford a compound 3 containing a saturated side chain. The antiparasitic assays performed with compound 3, acetylated (3a), and eliminated (3b) derivatives confirmed the lack of activity. Furthermore, an in-silico study using the SwissADME online platform was performed to bioactive compounds 1, 1a, and 1b in order to investigate their physicochemical parameters, pharmacokinetics, and drug-likeness. Despite the reduced effect against amastigote forms of the parasite to the purified compounds, different mixtures of compounds 1 + 2, 1a + 2a, and 1b + 2b were prepared and exhibited IC50 values ranging from 7.9 to 38.4 µM, with no toxicity for NCTC mammalian cells (CC50 > 200 µM). Selectivity indexes to these mixtures ranged from >5.2 to >25.3. The obtained results indicate that seeds of Porcelia macrocarpa are a promising source of interesting prototypes for further modifications aiming at the discovery of new antileishmanial drugs.

Optimization of physicochemical properties is a strategy to improve drug-likeness associated with activity: Novel active and selective compounds against Trypanosoma cruzi.

Trypanosoma cruzi is the causing agent of Chagas disease, a parasitic infection without efficient treatment for chronic patients. Despite the efforts, no new drugs have been approved for this disease in the last 60 years. Molecular modifications based on a natural product led to the development of a series of compounds (LINS03 series) with promising antitrypanosomal activity, however previous chemometric analysis revealed a significant impact of excessive lipophilicity and low aqueous solubility on potency of amine and amide derivatives. Therefore, this work reports different modifications in the core structure to achieve adequate balance of the physicochemical properties along with biological activity. A set of 34 analogues were designed considering predicted properties related to lipophilicity/hydrosolubility and synthesized to assess their activity and selective toxicity towards the parasite. Results showed that this strategy contributed to improve the drug-likeness of the series while considerable impacts on potency were observed. The rational analysis of the obtained data led to the identification of seven active piperazine amides (28-34, IC50 8.7 to 35.3 µM against intracellular amastigotes), devoid of significant cytotoxicity to mammalian cells. The addition of water-solubilizing groups and privileged substructures such as piperazines improved the physicochemical properties and overall drug-likeness of these compounds, increased potency and maintained selectivity towards the parasite. The obtained results brought important structure-activity relationship (SAR) data and new lead structures for further modifications were identified to achieve improved antitrypanosoma compounds.

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.

Calanolides E1 and E2, two related coumarins from Calophyllum brasiliense Cambess. (Clusiaceae), displayed in vitro activity against amastigote forms of Trypanosoma cruzi and Leishmania infantum.

In the present work, the MeOH extract from stem barks of Calophyllum brasiliense Cambess. (Clusiaceae) displayed activity against amastigote forms of Trypanosoma cruzi and Leishmania infantum and was subjected to a bioactivity-guided fractionation to give two related coumarins - calanolides E1 (1) and E2 (2). Compounds 1 and 2 were actives to T. cruzi with EC50 values of 12.1 and 8.2 µM, respectively. When tested against L. infantum, the EC50 values were 37.1 and 29.1 µM, respectively. Compound 2, corresponding to anti isomer, showed the best selectivity index (SI) with values >24.4 to T. cruzi and >6.9 to L. infantum in comparison to the syn isomer 1. Furthermore, using an in silico multi-parametric prediction, both compounds did not contain any PAINS sub-structures. Therefore, these data suggest that coumarins 1 and 2 may contribute as scaffolds for the design of novel drug candidates for leishmaniasis and Chagas disease.

Synthesis and Structure-Activity Relationship of Dehydrodieugenol B Neolignans against Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects over seven million people, especially in developing countries. Undesirable side effects are frequently associated with current therapies, which are typically ineffective in the treatment of all stages of the disease. Here, we report the first synthesis of the neolignan dehydrodieugenol B, a natural product recently shown to exhibit activity against T. cruzi. Using this strategy, a series of synthetic analogues were prepared to explore structure-activity relationships. The in vitro antiparasitic activities of these analogues revealed a wide tolerance of modifications and substituent deletions, with maintained or improved bioactivities against the amastigote forms of the parasite (50% inhibitory concentration (IC50) of 4-63 µM) and no mammalian toxicity (50% cytotoxic concentration (CC50) of >200 µM). Five of these analogues meet the Drugs for Neglected Disease Initiative (DNDi) "hit criteria" for Chagas disease. This work has enabled the identification of key structural features of the natural product and sites where scaffold modification is tolerated.

Sertraline Delivered in Phosphatidylserine Liposomes Is Effective in an Experimental Model of Visceral Leishmaniasis.

Liposomes containing phosphatidylserine (PS) has been used for the delivery of drugs into the intramacrophage milieu. Leishmania (L.) infantum parasites live inside macrophages and cause a fatal and neglected viscerotropic disease, with a toxic treatment. Sertraline was studied as a free formulation (SERT) and also entrapped into phosphatidylserine liposomes (LP-SERT) against intracellular amastigotes and in a murine model of visceral leishmaniasis. LP-SERT showed a potent activity against intracellular amastigotes with an EC50 value of 2.5 µM. The in vivo efficacy of SERT demonstrated a therapeutic failure. However, when entrapped into negatively charged liposomes (-58 mV) of 125 nm, it significantly reduced the parasite burden in the mice liver by 89% at 1 mg/kg, reducing the serum levels of the cytokine IL-6 and upregulating the levels of the chemokine MCP-1. Histopathological studies demonstrated the presence of an inflammatory infiltrate with the development of granulomas in the liver, suggesting the resolution of the infection in the treated group. Delivery studies showed fluorescent-labeled LP-SERT in the liver and spleen of mice even after 48 h of administration. This study demonstrates the efficacy of PS liposomes containing sertraline in experimental VL. Considering the urgent need for VL treatments, the repurposing approach of SERT could be a promising alternative.

Antitrypanosomal Activity of Acetogenins Isolated from the Seeds of Porcelia macrocarpa Is Associated with Alterations in Both Plasma Membrane Electric Potential and Mitochondrial Membrane Potential.

As part of a drug discovery program aimed at the identification of anti- Trypanosoma cruzi metabolites from Brazilian flora, four acetogenins (1-4) were isolated from the seeds of Porcelia macrocarpa and were identified by NMR spectroscopy and HRESIMS. The new compounds 1 and 2 displayed activity against the trypomastigote (IC50 = 0.4 and 3.6 µM) and amastigote (IC50 = 23.0 and 27.7 µM) forms. The structurally related known compound 3 showed less potency to the amastigotes, with an IC50 value of 58 µM, while the known compound 4 was inactive. To evaluate the potential mechanisms for parasite death, parameters were evaluated by fluorometric assays: (i) plasma membrane permeability, (ii) plasma membrane electric potential (ΔΨp), (iii) reactive oxygen species production, and (iv) mitochondrial membrane potential (ΔΨm). The results obtained indicated that compounds 1 and 2 depolarize plasma membranes, affecting ΔΨp and ΔΨm and contributing to the observed cellular damage and disturbing the bioenergetic system. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were nonmutagenic, noncarcinogenic, nongenotoxic, and weak hERG blockers. Additionally, none of the isolated acetogenins 1-4 were predicted as pan-assay interference compounds.

Antitrypanosomal activity of isololiolide isolated from the marine hydroid Macrorhynchia philippina (Cnidaria, Hydrozoa).

Marine invertebrates are a rich source of small antiparasitic compounds. Among them, Macrorhynchia philippina is a chemically underexplored marine cnidarian. In the search for candidates against the neglected protozoan Chagas disease, we performed a bio-guided fractionation to obtain active compounds. The structural characterization of the active compound was determined using NMR analysis and MS and resulted in the isololiolide, a compound described for the first time in this species. It showed in vitro activity against both trypomastigote and intracellular amastigotes of Trypanosoma cruzi, with IC50 values of 32 µM and 40 µM, respectively, with no mammalian cytotoxicity (>200 µM). The lethal action was investigated in T. cruzi using different fluorophores to study: (i) mitochondrial membrane potential; (ii) plasma membrane potential and (iii) plasma membrane permeability. Our results demonstrated that isololiolide caused disruption of the plasma membrane integrity and a strong depolarization of the mitochondrial membrane potential, rapidly leading the parasite to death. Despite being considered a possible covalent inhibitor, safety in silico studies of isololiolide also considered neither mutagenic nor genotoxic potential. Additionally, isololiolide showed no resemblance to interference compounds (PAINS), and it succeeded in most filters for drug-likeness. Isololiolide is a promising candidate for future optimization against Chagas disease.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study.

BACKGROUND: Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. METHODS: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. RESULTS: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. CONCLUSIONS: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study

Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)