Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 μM

Inhibition distribution of the <i>in vitro</i> screenings.

<p>The number of compounds is plotted against the percentage of TR inhibition. The grey area represents the activity distribution of the compounds in the initial screening, while the white area shows the distribution of the second <i>in vitro</i> screening based on the <i>in silico</i> enriched focused data set. The overall activity of the <i>in silico</i> enriched data set is shifted to the right compared to the initial diverse <i>in vitro</i> screen data set. Importantly, the second screen delivered more <i>in vitro</i> hits with an inhibitory potency of ≥30% although this data set was 3 times smaller.</p

TR-catalyzed reduction of trypanothione disulfide (TS₂) to the dithiol trypanothione (T(SH)₂).

<p>TR-catalyzed reduction of trypanothione disulfide (TS₂) to the dithiol trypanothione (T(SH)₂).</p

Effect of three top hits on the proliferation of <i>T</i>. <i>brucei</i>.

<p>Bloodstream form trypanosomes were cultured in the presence or absence of the respective compounds. After 48 h (light grey) and 72 h (grey), living cells were counted. The efficacy describes the inhibition of the cell proliferation in the presence of inhibitor compared to DMSO. Chlorhexidine served as positive control. The values are the mean ± SD from three independent series of experiments.</p

Evaluation of different binding models and corresponding K_i values.

<p>The Enzyme Kinetics Module from SigmaPlot 10 was used to evaluate the inhibition type and K_i values. A detailed statistical report was created for each binding model to compare different models and to determine the quality of fit to each model.</p><p>Evaluation of different binding models and corresponding K_i values.</p

Lineweaver-Burk plots for the most active compounds assuming an uncompetitive mode of inhibition.

<p>The K_<b>i</b> values represent the mean of two independent experiments; standard deviations are based on fitted plots. Data analysis has been performed using the Enzyme Kinetics Module of SigmaPlot, which fits the experimental data to the selected binding model.</p

TR assay validation—Lineweaver-Burk plots for known inhibitors.

<p>The K_<b>i</b> values were determined by three independent experiments. <b>Mepacrine:</b> measured Ki 20.71 ± 5.47 μM, competitive binding mode; <b>BG237:</b> measured Ki 42.13 ± 4.38 μM, noncompetitive binding mode, partial. The factor beta reflects the modification of the rate of product formation by the enzyme that is caused by the inhibitor. <b>Chlorhexidine:</b> measured Ki 6.13 ± 1.65 μM, competitive binding mode. Graphs are created using the SigmaPlot Enzyme Kinetics Module routine based on the relevant binding models and the calculated parameter values.</p

Schematic representation of the combined <i>in vitro</i> and <i>in silico</i> screening approach.

<p>After screening the highly diverse compound set of 2,816 compounds, 21 hits were obtained. These actives were then used as query for an <i>in silico</i> structure similarity search with the aim to create an activity enriched compound set. The resulting focused library of 1,204 compounds was screened again leading to additional 61 novel compounds with inhibitory activity against TR. Four out of the 82 combined, novel TR inhibitors showed activities of < 1 μM and were tested for their ability to interfere with the proliferation of cultured bloodstream <i>T</i>. <i>brucei</i>. Finally, three compounds showed activity in cell culture and were selected for further optimization efforts.</p

Calculated physicochemical properties and ADME parameters.

<p>¹⁾ Pipeline Pilot [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003773#pntd.0003773.ref032" target="_blank">32</a>]</p><p>²⁾ VolSurf+ [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003773#pntd.0003773.ref036" target="_blank">36</a>]</p><p>Calculated physicochemical properties and ADME parameters.</p

TR assay validation—measured versus published kinetic data.

<p>K_i ± SD (n = 3); IC₅₀ ± SD (n = 3)</p><p>TR assay validation—measured versus published kinetic data.</p