A Fluorinated Phenylbenzothiazole Arrests the Trypanosoma cruzi Cell Cycle and Diminishes the Infection of Mammalian Host Cells.

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells.

Trypanosoma cruzi is the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti-T. cruzi activity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC50) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K1 cells, exhibiting a 50% cytotoxic concentration (CC50) of 13.47 ± 0.37 µM and an IC50 of 0.14 ± 0.12 µM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 µM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (<90). These data suggest that MB17 could be an interesting lead compound against T. cruzi.

An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection.

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6-8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 "chimeric" molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 µM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 µM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

ATP regulates the activity of an alternative oxidase in Trypanosoma brucei.

The reduced mitochondrial respiratory chain from the bloodstream forms of Trypanosoma brucei is composed of only a membrane-bound glycerol-3-phosphate dehydrogenase and an alternative oxidase. Since these enzymes are not proton pumps, their functions are restricted to the maintenance of the redox balance in the glycosome by means of the dihydroxyacetone phosphate/glycerol-3-phosphate shuttle. Additionally, an F1 Fo -ATP synthase functions as an ATP-hydrolysing enzyme to establish the proton motive force necessary to maintain the basic functions of mitochondria. In this report, we studied the interplay between the alternative oxidase and ATP synthase, and we found that, in addition to its role as a proton pump, ATP synthase contributes to maintain safe levels of ATP to prevent the inhibition of the alternative oxidase by ATP.

Uptake of l-Alanine and Its Distinct Roles in the Bioenergetics of Trypanosoma cruzi.

Amino acids participate in several critical processes in the biology of trypanosomatids, such as osmoregulation, cell differentiation, and host cell invasion. Some of them provide reducing power for mitochondrial ATP synthesis. It was previously shown that alanine, which is formed mainly by the amination of pyruvate, is a metabolic end product formed when parasites are replicating in a medium rich in glucose and amino acids. It was shown as well that this amino acid can also be used for the regulation of cell volume and resistance to osmotic stress. In this work, we demonstrate that, despite it being an end product of its metabolism, Trypanosoma cruzi can take up and metabolize l-Ala through a low-specificity nonstereoselective active transport system. The uptake was dependent on the temperature in the range between 10 and 40°C, which allowed us to calculate an activation energy of 66.4 kJ/mol and estimate the number of transporters per cell at ~436,000. We show as well that, once taken up by the cells, l-Ala can be completely oxidized to CO2, supplying electrons to the electron transport chain, maintaining the electrochemical proton gradient across the mitochondrial inner membrane, and supporting ATP synthesis in T. cruzi epimastigotes. Our data demonstrate a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.IMPORTANCE It is well known that trypanosomatids such as the etiological agent of Chagas' disease, Trypanosoma cruzi, produce alanine as a main end product of their energy metabolism when they grow in a medium containing glucose and amino acids. In this work, we investigated if under starvation conditions (which happen during the parasite life cycle) the secreted alanine could be recovered from the extracellular medium and used as an energy source. Herein we show that indeed, in parasites submitted to metabolic stress, this metabolite can be taken up and used as an energy source for ATP synthesis, allowing the parasite to extend its survival under starvation conditions. The obtained results point to a dual role for Ala in the parasite's bioenergetics, by being a secreted end product of glucose catabolism and taken up as nutrient for oxidative mitochondrial metabolism.