Fig 1.
Validation of crystal violet as proline transporter TcAAAP069 inhibitor.
(a) Inhibition of proline transport in wild type parasites (TcWT, black circles) and parasites overexpressing TcAAAP069 (Tc069, white squares). (b) Trypanocidal effect of crystal violet in TcWT (black circles) and Tc069 (white squares) parasites. The data is expressed as the mean ± standard deviation and corresponds to three independent experiments.
Fig 2.
Chemical structures of crystal violet, its analogues and negative control.
The figure shows the chemical structure of crystal violet (a), the structure of the negative control, dapsone, (b) and the structures of the four crystal violet structural analogues loratadine, cyproheptadine, olanzapine and clofazimine (c).
Fig 3.
Binding poses suggested by molecular docking between proline, crystal violet and its chemical analogues with proline permease TcAAAP069.
(a) Homology modelled TcAAAP069 (represented as cartoon) highlighting the predicted interaction sites of proline (PRO site, green) and crystal violet (CV site, violet; and dark shadowed-violet indicates the hydrophobic “hot-spot”). The predicted binding poses of proline (red), CV (violet), loratadine (blue), cyproheptadine (yellow), olanzapine (dark-grey), clofazimine (green) and dapsone (pink) are shown in the figure. (b) Detail of the residues predicted to interact with CV (right side) and proline (left side).
Table 1.
Compounds obtained by similarity screening and molecular docking analysis.
Fig 4.
Calculation of CV structural analogues concentrations required to inhibit 50% of proline transport activity (IC50).
The IC50s were calculated for three of the four CV analogues. It was not possible to calculate an IC50 for OLZ since this compound produced the death of parasites in the lapse time of the assay in concentrations over 300 μM. The data is expressed as the mean ± standard deviation and corresponds to three independent experiments. CV, crystal violet. LTD, loratadine. CPH, cyproheptadine. OLZ, olanzapine. CFZ, clofazimine. N/A, not available.
Table 2.
Trypanocidal effect of CV chemical analogues in T. cruzi Y strain.
Table 3.
Trypanocidal effect (IC50, μM) of CV structural analogues in epimastigotes from different strains of T. cruzi.
Fig 5.
Synergism between benznidazole and the combination of the crystal violet analogues in T. cruzi trypomastigotes.
(a) Drug combinations between BZL and CV analogues (LTD-CPH-CFZ). Combination index (CI) value for each combination point is presented under the corresponding graded symbol. Graded symbols mean strong synergism (++++, CI between 0.1–0.3), synergism (+++, CI between 0.3–0.7), moderate synergism (++, CI between 0.7–0.85) and nearly additive effect (±, CI between 0.9–1.1). The boxes coloured with light-grey correspond to the combination points where no synergism was observed. (b) Chou-Talalay plot. Representation of CI vs effect (Fa, fraction affected), where CI > 1, CI = 1 (dotted line) and CI < 1 indicate antagonism, additive effect and synergism, respectively. For each data series BZL concentrations increase from left to right (from 0.5 to 10 μM). The data is expressed as the mean ± standard deviation of three independent experiments. All calculations were performed with CompuSyn software. LTD, loratadine. CPH, cyproheptadine. CFZ, clofazimine. BZL, benznidazole. IC50 LTD = 15 μM. IC50 CPH = 10 μM. IC50 CFZ = 5 μM. LTD-CPH-CFZ, combination of the three crystal violet analogues as a single drug. 1/3 IC50, refers to the sum of 1/3 of each IC50, 5 μM + 3.67 μM + 1.33 μM = 10 μM. 1/4 IC50, 3.75 μM + 2.5 μM + 1.25 μM = 7.5 μM. 1/5 IC50, 3 μM + 2 μM + 1 μM = 6 μM. 1/10 IC50, 1.5 μM + 1 μM + 0.5 μM = 3 μM.