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.

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.

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.

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.