Efficacy of nifurtimox, benznidazole and posaconazole against <i>T</i>. <i>cruzi</i> Silvio X10/7 and Tulahuen βgal trypomastigotes at 72 h.

<p>pEC₅₀ = –Log (EC₅₀ [M]), average of at least three biological replicates ± SD. Additional data for 24 h and 48 h time points available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006612#pntd.0006612.s014" target="_blank">S5 Table</a>.</p

Model to explain the effect of starting-density on the dose-response profile.

<p>Measured (solid lines, 0% inhibition, growth rate = 1) and predicted (dashed lines) growth curves for <i>T. brucei</i> in the standard (STD) and static-cidal (SC) assays. Predicted growth curves are for 50% inhibition of growth rate (long dashes, growth rate = 0.5) and 75% inhibition of growth rate (short dashes, growth rate = 0.25). Percent inhibition as calculated at t = 72 h (STD) or t = 48 h (SC) is given on right hand side. The yellow shaded area represents cell densities above the detection limit.</p

Potencies against intracellular <i>T</i>. <i>cruzi</i> for control compounds (N = 3).

<p>Potencies against intracellular <i>T</i>. <i>cruzi</i> for control compounds (N = 3).</p

Effect of the assay protocol on the dose-response profile.

<p>A: In the standard assay (left) compound DDU1 displays monophasic behaviour, while a biphasic fit is obtained in the static-cidal assay (right). B: Melarsoprol shows monophasic behaviour in both the standard assay (left) and the static-cidal assay (right).</p

Growth curves obtained in the static-cidal assay for control compounds and representative test compound.

<p>Growth curves are shown for each compound, with the concentration of compound indicated on the right hand side of each growth-line (µM). MCCs are indicated for each compound. All measurements are the average of three independent experiments. The error bars indicate the standard deviation. RFU = relative fluorescence units.</p

Analysis of the hits.

<p>Panel A. All the hits from the 5 μM screen were annotated with a general pharmaceutical class. The chart shows the frequency for each class. Panel B. The potencies for all the hits were determined and represented as a frequency table. Bars in red represent CYP51 inhibitors, green = all other classes).</p

<i>T</i>. <i>cruzi</i> rate-of-kill assay.

<p>Panel A. Outline of the rate-of-kill assay. Numbers are time in hours. Times above the timeline are from addition of the trypomastigotes, times below the line are starting from addition of the infected cells to the plates containing compounds. Panel B. Representative images from untreated wells in the static-cidal assay at the different time-points. Panel C. Rate-of-kill profiles. Left panel shows percent infected cells against time, right panel shows Vero counts against time (n = 3, error bars = standard deviation) for Nifurtimox (top), Benznidazole (middle) and Posaconazole (bottom). Concentrations are as follows (μM) (all compounds were tested at the same concentrations except for Posaconazole, its concentrations are shown between brackets): black circle 50 (1), red triangle 17 (0.3), green square 5.6 (0.1), yellow diamond 1.9 (0.04), blue triangle 0.6 (0.01), pink hexagon 0.2 (0.004), cyan circle 0.07 (0.001), triangle dark yellow 0.</p

Drug efficacy against <i>T</i>. <i>cruzi</i> intracellular amastigotes.

<p>Strains Silvio X10/7, Y and PAH1790 assessed after 5 days treatment with nifurtimox <b>(A)</b> benznidazole <b>(B)</b> and posaconazole <b>(C)</b>. (Representative dose-response curves of at least 3 biological replicates normalised to 0% effect DMSO and 100% effect 50 μM nifurtimox controls. Average±SD). The x axis is the Log of the compound concentration in Molar.</p

Drug efficacy against <i>T</i>. <i>cruzi</i> panel strains at 120 h.

<p>pEC₅₀ = –Log (EC₅₀ [M]), average of at least three biological replicates ± SD. Additional data for 72 h and 96 h time points available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006612#pntd.0006612.s012" target="_blank">S3</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006612#pntd.0006612.s013" target="_blank">S4</a> Tables respectively.</p

<i>T</i>. <i>cruz</i>i strain panel encompassing the six major <i>T</i>. <i>cruzi</i> discrete typing units (DTU’s) TcI-VI.

<p><i>T</i>. <i>cruz</i>i strain panel encompassing the six major <i>T</i>. <i>cruzi</i> discrete typing units (DTU’s) TcI-VI.</p