Fig. 4.
Chemical targeting of TRYS in T. brucei. A. Chemical structure of TRYS inhibitor DDU 86439 (N-(3-(dimethylamino)propyl)-2-(3-(3-fluorophenyl)-1H-indazol-1-yl)acetamide). B. EC50 values were determined for DDU 86439 against WT (closed circles), SKO (open squares), TRYS-overexpressing (open circles) and cDKO cells grown in the absence of tetracycline for 2 days prior to analysis (closed squares, dotted line). The curves are the non-linear fits of data using a two-parameter EC50 equation provided by GraFit (see Experimental procedures). EC50 values of 7.1 ± 0.2, 1.2 ± 0.05, 23.3 ± 0.3 and 0.46 ± 0.01 μM were determined for WT, SKO, TRYS-overexpressing and cDKO cell lines respectively. Data are the mean of duplicate measurements. C. Analysis of intracellular T[SH]2 (closed circles) and GSH (open circles) levels in T. brucei bloodstream parasites (WT) following incubation with compound DDU 86439 (72 h). Data are represented as a percentage of the thiol levels of untreated cells (0.51 and 0.42 nmol(108 cells)−1 for GSH and T[SH]2 respectively; Table S1) and each measurement is the mean of three individual measurements.