Figure 1.
Chemical structures of compounds used in this study.
The title compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (1) and its amino substituted derivatives 4-[4-(5-morpholine-4-yl-1H-pyrazol-3-yl)-phenoxy]phenylamine (2), 4-[5-morpholine-4-yl-3-(4-phenoxyphenyl)-pyrazol-1-yl]phenylamine (3) and 1-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]piperazine (4) are presented. 3-(4-phenoxyphenyl)-1H-pyrazole (5) showed very low antiparasitic activity (IC50 of >65 µM) as well as general toxicity (IC50 of >136 µM), therefore it was used as negative control for the biochemical, biophysical and compound sensitivity tests.
Figure 2.
SDS-PAGE (12%) analysis of proteins retained by the affinity matrices.
Panel A: Matrix linked with derivative 2. Panel B: Matrix linked with derivative 3. Panel C: Matrix linked with derivative 4. Samples of T. b. rhodesiense lysate (200 µg total protein content) were incubated with either 40 µl control beads (lanes a) or 40 µl affinity beads (lanes b). Proteins retained by the matrices were directly separated by SDS-PAGE. Arrows indicate protein bands resulting from the compound derived matrices. AK: adenosine kinase of T. b. rhodesiense; GAPDH: glycosomal glyceraldehyde-3-phosphate dehydrogenase of T. b. rhodesiense; K: human keratin. Molecular mass markers are shown on the left.
Figure 3.
Concentration dependence of the activation effect for compounds 1 to 5 in the range of 1–750 µM.
Increasing concentrations of compound 1 (•) gives a sigmoid saturation curve for TbrAK activation, yielding an EC50 value of 38.9±0.9 µM. A similar trend is observed for compound 2 (▴), but due to limited solubility at concentrations >50 µM the EC50 could not be determined. Compound 4 (▪) started activating TbrAK in a concentration dependent manner without reaching a maximum at the solubility limiting concentration of 750 µM. Compound 3 (♦) and the control compound 5 (★) did not activate TbrAK under identical conditions. Values are reported as % activity derived from the transformation rate. For comparative reasons the activity recorded in absence of compound was set to 100%. Standard deviations are represented by vertical bars. The mean of three independent measurements is reported.
Table 1.
Thermal stability assay regarding TbrAK in absence and presence of compounds and substrates.
Figure 4.
Specific binding of compound 1 to TbrAK measured by ITC.
The top panel shows heat signals upon 27 injections of compound 1 (trace I) or control compound 5 (trace II) into the sample cell containing 7 µM TbrAK. The binding isotherm obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution is shown on the lower panel. The solid line represents the non-linear least square fit based on a two-sites non-interacting binding model. Compound 1 binds to TbrAK via a high affinity binding site with a KD of 75±20 nM and a ΔHbind of −3.05±0.77 kcal/mol, and a low affinity site exhibiting a KD of 497±34 nM and a ΔHbind of −1.13±0.24 kcal/mol. No specific heat release was shown for the negative control (trace II). The mean of three independent experiments are reported.
Figure 5.
Representative plots for TbrAK kinetics (AMP formation) with respect to adenosine in absence (○) and presence (•) of compound 1 (33 µM).
Adenosine strongly inhibits TbrAK at concentrations >2 µM and follows typical substrate-inhibition kinetics. In contrast, compound 1 abolishes substrate-inhibition. The solid lines indicate the fit of the raw data with the substrate-inhibition model (see Table 2 for fitting results).
Table 2.
Parameters derived from TbrAK kinetics with respect to adenosine in absence and presence of compound 1.
Figure 6.
Dose-response curve for parasite growth in presence of compound 1.
RNAi-mediated silencing of TbAK expression by tetracycline (•) reduces sensitivity of TbAK RNAi cells to compound 1 when compared to non-induced cells (○), raising the IC50 from 131±43 nM to 271±25 nM (two-tailed t-test, p<0.05).