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

Abstract

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

“…The cells were labelled, or not for the unstained control, by the addition of 256 nM Rhodamine 123 (Rho123) for 20 min at 28°C. The cells were twice in washed with cytomix buffer (25 mM HEPES-KOH, 120 mM KCl, 0.15 mM CaCl 2 , 2 mM EDTA, 5 mM MgCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 buffer, pH 7.6, and 10 μM FCCP if indicated) and resuspended in 500 μL of the same buffer [ 31 ]. Changes in the cell’s fluorescence labelled with Rho123 were analysed by flow cytometry on a BD Accuri™ C6 Plus.…”

“…Then, the cells (1.0x10 7 cells/mL) were collected by centrifugation (2,700 x g for 5 min), washed in PBS and fixed in 70% ethanol for 4 h. The parasites were washed twice in PBS and incubated with 10 μg/mL RNase A (Thermo Scientific) for 30 min at 37°C. To measure the DNA content, the cells were stained with 40 μg/mL propidium iodide (Molecular Probes/Invitrogen) and analysed by flow cytometry on a BD Accuri™ C6 Plus, with 50,000 events collected from three biological independent experiments [ 31 ]. Histograms (number of counts by FL2 area), scatter plots (side scatter [SSC] area by forward scatter [FSC] area) and gates for each cell cycle phase were analysed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v07).…”

“…It was shown that these compounds preferentially bind to the kDNA and interfere with mitochondrial functions and consequently with the proliferation of parasites [18]. This is not surprising, since it has been reported that T. cruzi can survive for long periods of time without triggering any cell death mechanism after having its proliferation arrested [18,31,41].…”

“…Plus, with 50,000 events collected from three biological independent experiments [31]. Histograms (number of counts by FL2 area), scatter plots (side scatter [SSC] area by forward scatter [FSC] area) and gates for each cell cycle phase were analysed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v07).…”

Synthesis, antiproliferative and antitrypanosomal activities, and DNA binding of novel 6-amidino-2-arylbenzothiazoles

“…The drugs currently used to treat HAT are not effective against all stages and subspecies of the parasite, so further clinical investigation is needed to develop new antitrypanosomal drugs. Some 2-benzylsulfanyl- and 2-arylbenzothiazole derivatives have been found to exhibit good trypanocidal activity at low concentrations 35 , 36 . Optimisation of anti-parasite activity, physicochemical parameters and ADME properties afforded the fluoro-substituted benzothiazole, with a 2-cyclopropanecarboxamide at position 2, which displayed promising in vivo efficacy 37 .…”