Fig 1.
Quinone amounts in HeLa cell cultures treated with compound 1.
HeLa cells (4 × 105 cells/well) were cultured in the presence of compound 1 for 2 days at the indicated concentrations, and the amounts of UQ10 and DMQ10 in the harvested cells were analyzed. The graph is representative of two independent experiments performed in duplicate quantification. The quinone amounts (ng) / well are presented as mean ± standard deviation.
Fig 2.
Growth of T. cruzi epimastigotes treated with compound 1 and BNZ.
The luciferase-expressing epimastigotes (5 × 105 cells/ml) were cultured for 4 days in a medium containing various concentrations of the compounds, and their numbers were determined by measuring the luciferase activity. The open circles indicate the results of BNZ treatment, and the closed circles indicate those of compound 1. The typical results of three independent experiments are shown. Epimastigote growth was determined by triplicate quantification and is indicated as mean ± standard deviation.
Table 1.
List of the oxazinoquinoline derivatives effective on epimastigote growth.
Fig 3.
Inhibition of epimastigote growth by compounds 1–3.
Dose-response effects of the compounds on epimastigote growth after a 4-day treatment are shown. The regression curves and EC50 values were calculated by biphasic non-linear regression using the software PRISM 8.4.1. The graph is representative of three independent experiments and the growth rates (%) are indicated as mean ± standard deviation.
Fig 4.
Inhibition of T. cruzi infection by compounds 1–3.
Compounds were added to infected host cultures at the indicated concentrations. After 4 days, the infection rates were manually counted using three photos showing 235–607 total host cells and inhibition rates were calculated. The graph is representative of two independent experiments and indicates mean inhibition ± standard deviation.
Table 2.
Effects of compounds on amastigote infection and human culture cells.
Fig 5.
Quinone amounts in HeLa cells treated with compounds 1, 2, and 3.
HeLa cells (2 × 105 cells/well) were inoculated and pre-cultured for 1 day, and cultured for further 2 days in the presence of the compounds at the indicated concentrations. The amounts of UQ10 and DMQ10 (ng) / protein (mg) were determined and normalized with the UQ10 amount of each control culture. The gray bar indicates the value of UQ10, and the black bar indicates that of DMQ10. The typical results of three independent experiments are shown with mean ± standard deviation of duplicate results.
Fig 6.
(A) The mass spectrum and (B) tandem mass spectrum of UQ9 contained in the epimastigote quinone fraction.
Fig 7.
Quinone components of epimastigotes treated with or without compound 1.
Representative chromatograms of quinone fractions from (A) control epimastigotes, (B) epimastigotes treated with 1 μM compound 1 for 4 days, and (C) RAW264.7 cells treated with 20 μM CQ for 1 day.
Fig 8.
Quinone amounts in epimastigotes treated with compound 1.
Quinone fractions were extracted from epimastigotes (1 × 107 cells) treated with various concentrations of compound 1 for 4 days. The epimastigote growth (%) in each culture was calculated (white bars). The amounts of UQ9 (gray bars) and DMQ9 (black bars) were calculated by comparing their signal areas to that of the added internal standard, UQ10. The mean quinone amount (ng) / 107 cells are indicated as mean ± standard deviation of duplicate measurements.
Fig 9.
Addition of UQ10 weakened the trypanocidal activity of the oxazinoquinoline derivatives.
Dose-response effects of the compounds on luciferase-expressing epimastigote growth were measured after a 4-day culture using the medium with (closed circles) or without (open circles) 30 μM UQ10. The typical results of two independent experiments are shown. Growth (%) is indicated as mean ± standard deviation of triplicate results.