https://orcid.org/0000-0002-3018-060X
Figures & data
Figure 1. Percentage of viability of T. cruzi epimastigotes and cellular TIM activity. 1 × 106 epimastigotes were incubated for 4 h at 28 °C with increasing concentrations of Rbz. Viability was subsequently determined using MTT (A), and the cellular activity of the glycolytic enzyme TIM, was also measured (B). Results represent the average of four independent biological experiments.
Figure 2. Cell viability and enzymatic activity of TIM from T. cruzi epimastigotes challenged with Rbz. Different number of T. cruzi epimastigotes were incubated with 250 µM Rbz for 24 h at 28 °C. The plots show the percentage of cell viability determined using MTT (A) and cellular TIM activity of protein extracts from trypanosomes (B). Results represent the average of four independent biological experiments.
Figure 3. Cell viability and enzymatic activity of cellular TIM from T. cruzi epimastigotes challenged with Rbz. Different number of T. cruzi epimastigotes were incubated with 1 mM Rbz for 4 h at 28 °C. The plots show the percentage of cell viability determined using MTT (A) and the cellular TIM activity of protein extracts from trypanosomes (B). Results represent the average of four independent biological experiments.
Figure 4. Quantification of MGO and AGEs in T. cruzi epimastigotes treated with Rbz. 1X106 epimastigotes were incubated with increasing concentrations of Rbz for 4 h at 28 °C. (A) Concentration of MGO/million cells at increasing concentrations of Rbz. (B) Concentration of AGEs in µg/mL per million cells at increasing concentrations of Rbz. Both metabolites were determined from protein extracts of epimastigotes incubated with different concentrations of Rbz. Results represent the average of four independent biological experiments.
Figure 5. Effect of Rbz on the enzymatic activity of TcTIM and HsTIM. 0.2 mg/mL of the recombinant enzymes were exposed to increasing concentrations of Rbz for 2 h at 37 °C. Subsequently, an aliquot was taken, and the enzyme activity was measured in a coupled assay. Red squares correspond to TcTIM and green circles to HsTIM. Results represent the average of four independent biological experiments.
Figure 6. Circular Dichroism spectra of TcTIM. Protein was prepared for the experimental conditions, in (A) green squares represent the enzyme incubated in absence of Rbz, whereas red circles represent the enzyme incubated in presence of Rbz. The Tm was also determined for these samples. In (B), open squares are the enzyme without Rbz (control) and open circles are the enzyme incubated in presence of Rbz. The red line corresponds to the Boltzmann sigmoid equation adjusted to the experimental data. Results represent the average of four independent biological experiments.
Table 1. Determination of the Cys content in the TcTIM in the absence and in presence of Rbz.
Figure 7. Effect of Rbz on the enzymatic activity of TcTIM WT and single mutants of Cys. All recombinant enzymes were incubated at 0.2 mg/mL and were exposed to increasing concentrations of the drug Rbz for 2 h at 37 °C. At the end of the incubation time, an aliquot was taken, and enzyme activity was measured by the coupled assay system as reported in the Material and Methods section. Black and orange squares correspond to WT and the C15A mutant, respectively; green and red circles correspond to the C118A and C40A, respectively; and the purple triangles correspond to the C127A mutant. Results represent the average of four independent biological experiments.
Table 2. Determination of the Cys content in TcTIM WT and single mutants of Cys in the absence and presence of Rbz.
Figure 8. Molecular docking of Rbz on TcTIM. The crystallographic structure of TcTIM (PDB code: 1tcd) and the 3D coordinates of Rbz were used for molecular docking using Docking Vina and the Blind Docking Server, as mentioned in the body of the text. (A) Blind docking of Rbz on the 3D structure of TcTIM. (B–D), Docking of Rbz at the cavities close to Cys 15, 40, and 118, respectively. (E) The scores obtained for the docking of Rbz at the interface and the cavities close to Cys 15, 40, and 118. The figures were modelled with PyMol Molecular Graphics System software (version 2.5.0, Schrödinger, LLC, New York, NY, USA).
Figure 9. Cartoon and stick representations of molecular docking in Rbz vs TcTIM. The crystallographic structures of (A) non-derivatized TcTIM (1tcd), and (B) derivatized-TcTIM with DTBA (2oma) were used with the 3D coordinates of the ligand Rbz; green and cyan colours represent each subunit of the dimeric protein. (C) The crystallographic structures of 1tcd (purple) and 2oma (gray) were superimposed obtaining an overall RMSD of 0.366 Å for C-α. Despite significant structural coincidence, loop 3 of 2oma is displaced 7.5 Å with respect to 1tcd, (zoom view in panel (C). The figures were modelled with PyMol Molecular Graphics System software (version 2.5.0, Schrödinger, LLC, New York, NY, USA).
