Chemical structures of the five lead compounds selected for <i>in vivo</i> studies.

<p>Chemical structures of the five lead compounds selected for <i>in vivo</i> studies.</p

Adult worm IC₅₀ values of five <i>in vivo</i> candidates over time post drug exposure.

<p>Values were determined 1, 2, 4, 7, 10, 24, 48 and 72>33.3 µM are indicated with (<b>//</b>). PZQ: praziquantel.</p

Worm burden reductions observed for the five lead candidates in <i>S. mansoni</i> infected mice.

<p>Mice harbored a patent <i>S. mansoni</i> infection. Different dosage regimens were used (1×400 mg/kg, 4×80 mg/kg on four consecutive days or 4×100 mg/kg every 4 hours).</p><p>WBR: Worm burden reduction.</p><p>p-value<0.05.</p><p>p-value<0.005.</p

Adult worm IC₅₀ and IC₉₀ values of five <i>in vivo</i> candidates compared to praziquantel 72 hours post drug exposure.

<p>PZQ: Praziquantel.</p

Screening flow.

<p>Screening was conducted at the Swiss TPH (screening cascade <b>I</b>; steps <b>A</b>–<b>E</b> and Quality control): Primary screening steps (yes/no filters) of 100 µM and 33.3 µM resulted in 179 and 72 hits, respectively. Active compounds (n = 72) moved on to Step <b>C</b> and IC₅₀ values were evaluated on NTS. Thirty-four compounds showed activities with IC₅₀ values <10 µM and pre-screening was conducted on adult schistosomes (Step <b>D</b>). Active compounds (n = 16) with schistosomicidal effects at 33 µM compound concentration were further characterized (step <b>E</b>). The quality control represents randomly selected compounds from compounds classified as non-active from the pre-screening steps (step <b>A</b>/<b>B</b>) on NTS which were re-evaluated in step <b>B</b>. In parallel, all compounds (n = 400) were studied at the LSHTM in London (screening cascade <b>II</b>): step <b>A</b>, all 400 compounds were screened on <i>S. mansoni</i> adults at 15 µM. Step <b>B: 44</b> compounds were active and these were then tested for IC₅₀ determination on adult worms. From both screening cascades, five compounds were selected for <i>in vivo</i> testing based on pharmacodynamic and pharmacokinetic properties as well as toxicity.</p

Characterization of five lead candidates selected for <i>in vivo</i> testing.

<p><i>In vitro</i> activity on <i>P. falciparum</i> 3D7, NTS, adult <i>S. mansoni</i>, cytotoxicity on MRC5-cells, and pharmacokinetic parameters* of 5 active compounds selected for <i>in vivo</i> studies identified in 2 parallel screens at the Swiss TPH and LSHTM.</p><p>PK parameters are unpublished data, <i>In vitro</i> activity on <i>P. falciparum</i> 3D7 and cytotoxicity on MRC5-cells can be found at <a href="http://www.mmv.org/research-development/malaria-box-results" target="_blank">http://www.mmv.org/research-development/malaria-box-results</a>.</p

Malaria Box Heatmap.

<p>Shown are selected data from the HeatMap (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s002" target="_blank">S1 Table</a>) for the 400 Malaria Box compounds. Each column represents an assay (grouped by category), compounds are represented in rows. The red-green gradient represents higher to lower activity. Favorable PK activities are scored green. <i>Pf</i>: <i>Plasmodium falciparum</i>, <i>Pb</i>: <i>Plasmodium berghei</i>, PK: pharmacokinetics, sol.: solubility, hERG: human ether-a-go-go channel inhibition, DDI: drug-drug interactions (predicted).</p

Metabolomic and chemogenomic profiling.

<p>(A) Metabolic profiling: Heat map showing metabolic fingerprints of 80 Malaria Box compounds and atovaquone control. Parasite extracts were analyzed by LC-MS, and changes in metabolite pools were calculated for drug-treated parasites as compared to untreated controls. Hierarchical clustering was performed on ²log-fold changes in metabolites (data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s003" target="_blank">S2 Table</a>), scaled from -3 to +3. Six of seven compounds (indicated in red) reported to target <i>Pf</i>ATP4 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.ref025" target="_blank">25</a>] showed a distinct metabolic response characterized by the accumulation of dNTPs and a decrease in hemoglobin-derived peptides. A large cluster of compounds (indicated in blue) clustered with the atovaquone control (indicated in orange), and exhibit an atovaquone-like signature characterized by dysregulation of pyrimidine biosynthesis, and showed a distinct metabolic response characterized by the accumulation of dNTPs and a decrease in hemoglobin-derived peptides. (B) Chemogenomic profiling: A collection of 35 <i>P</i>. <i>falciparum</i> single insertion <i>piggyBac</i> mutants were profiled with 53 MMV compounds and 3 artemisinin (ART) compounds [Artesunate (AS), Artelinic acid (AL) and Artemether (AM)] for changes in IC₅₀ relative to the wild-type parent NF54 (data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s004" target="_blank">S3 Table</a>, genes queried in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.s005" target="_blank">S4 Table</a>). Clone PB58 carried a <i>piggyBac</i> insertion in the promoter region of the K13 gene and has an increased sensitivity to ART compounds as do PB54 and PB55 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005763#ppat.1005763.ref033" target="_blank">33</a>]. Drug-drug relationships based on similarities in IC₅₀ deviations of compounds generated with <i>piggyBac</i> mutants created chemogenomic profiles used to define drug-drug relationships. The significance of similarity in MoA between Malaria Box compounds and ART was evaluated by Pearson’s correlation calculations from pairwise comparisons. The X axis shows the chemogenomic profile correlation between a Malaria Box compound and AS, the Y axis with AM; the color gradient indicates the average correlation with all ART derivatives tested. Five Malaria Box compounds (MMV006087, MMV006427, MMV020492, MMV665876, MMV396797) were identified as having similar drug-drug chemogenomic profiles to the ART sensitivity cluster.</p

Antiprotozoal Malaria Box compounds with activity in biological assays and lacking toxicity at therapeutic levels.

<p>Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like.</p