Fig 1.
Schematic representation of the trypanothione pathway.
Table 1.
Crystal parameters, data collection statistics and refinement statistics of compound 3-TR complex.
Fig 2.
(A) Schematic representation of the TR assay reaction. The luciferase activity is dependent on the residual NADPH concentration after TR reaction (1). The luminescent signal, which is produced by the NADPH-Glo assay, is inversely proportional to the TR activity (2). (B) Sensitivity and linearity of the NADPH detection. The plot x- and y-axis are presented in logarithmic scale to allow a better appreciation of the serially diluted points. The linear correlation is plotted as a sigmoid curve on the log-log graph. The plotted points are the average of three independent replicates.
Fig 3.
Optimization of the TR reaction.
(A) Titration of TS2 in the presence of 0.5 nM TR and 12.5 μM NADPH using the luminescent assay. (B) Titration of TS2 in the presence of 20 nM TR and 100 μM of NADPH using the DTNB assay. (C) TR activity time course and enzyme dilution in the presence of 12.5 μM NADPH and 15 μM TS2. (D) Dose-dependent inhibition of TR activity by auranofin (AF) as determined by the DTNB and the luminescence based assay. Each experimental point is the result of three replicates (error bars bigger than the symbol size were not plotted).
Fig 4.
(A). TR activity assay Z’ time course. All components were stored at 4°C during the time course with the exception of the NADPH-Glo reagent which was kept at 22°C. (B) Z′ distribution of all collection plates. (C) Compound activity distribution reported as number of standard deviations with respect to the whole sample average and standard deviation (error bars bigger than the symbol size were not plotted).
Fig 5.
General structures of the two major hit series (substructures).
Representative molecules for both series with TR inhibition potency data (examples).
Fig 6.
Compound 3 binding to TR as measured by SPR.
Every other four experimental point is plotted to allow a cleaner representation. The plot is one representative experiment of four.
Fig 7.
Compound 3 competition with either TS2 or NADPH for the TR activity.
Either TS2 or NADPH were titrated alone or against three compound concentrations (4, 16 and 64 μM). The NADPH concentration for the TS2 dilutions (A) was 40 μM while the TS2 concentration for the NADPH dilutions (B) was 30 μM. The TR assay was performed using 1 nM TR for 5 min. Each experimental point is the average of three replicates (error bars bigger than the symbol size were not plotted).
Fig 8.
(A) Overall fold of TR in complex with compound 3. The monomers A and B are colored in blue and magenta respectively. Compound 3 and FAD molecules are represented as sticks and colored cyan and magenta respectively. (B) Omit map (Fo-Fc) of the compound 3-TR complex active site. The map is colored green and contoured at 3σ. (C) Blow up of the ligand binding site. The residues interacting with the ligand are indicated and represented as sticks. The picture was obtained using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Fig 9.
(A) Superimposition between compound 3-TR complex and the apo TR (PDB code: 2JK6) structures. The structure of apo TR is colored blue, the compound 3-TR complex is colored magenta. The residues involved in the ligand binding are indicated and represented as sticks. (B) Superimposition between compound 3-TR complex (in magenta) and the reduced TR in complex with NADPH (in green) (PDB code: 2W0H). The two Arginine residues (Arg222 and Arg228) involved in the ligand binding are indicated and depicted as sticks. Compound 3 and NADPH are depicted as sticks and colored in light blue and green respectively (C) Superimposition between compound 3-TR complex (in magenta) and Glutathione reductase (in yellow) (PDB code: 3GRS). The residues involved in the ligand binding are indicated (the residues of the compound 3-TR complex on the left side and the residues of GR on the right side) and depicted as sticks. The picture was obtained using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Fig 10.
% Inhibition calculated on L. infantum promastigote stage after treatment with various concentrations of compound 3.
The data are expressed as mean ± standard error of two independent experiments.