All supplied compounds were assured by the vendor as 90% pure with quality control selleck chemicals llc data provided and were verified internally at SJCRH after plating. An initial search of the GlaxoSmithKline clinical development pipeline on a commercially available data base revealed around 100 compounds that had been taken into clinical development and subse quently been discontinued. Excluding those molecules that had already been screened against P. falciparum in the GSK discovery library, samples were obtained from GSK for 63 new compounds. GSK verified samples for purity and activity, and conducted the in vitro testing for this compound set. Pfizer Inc were also approached, and offered to screen their STLAR library of 176 drugs, comprised mainly of pre Phase III discontinued clinical candi dates, though Phase III data were available for a few compounds.

There were no approved drugs or active clinical candidates in the set. Pfizer provided samples verified for purity and activity. First, the compound set was tested in vitro using high throughput screen ing by Discovery Biology, Griffith University, Nathan, Australia with subsequent EC50 determination by Pfizer in house. AstraZeneca identified a set of 100 candidate drugs from other therapeutic areas for testing against P. falciparum. All 100 candidates had been discontinued for the original indication, and Phase I/II data were available for several compounds. AZ verified the samples for purity and conducted in vitro and in vivo testing for the compounds. None of the test sets described above was prescreened for pharmacokinetics/safety but included in their entirety.

This was because identification of any active compound could also have led to testing of related follow up com pounds that did not reach clinical testing. In vitro screening assays More detailed information on the in vitro methods is provided in Additional file 1. SJCRH used the SYBR I dye DNA staining assay, which measures proliferation of P. falciparum in human erythrocytes. Plasmodium falciparum strains 3D7 and K1 were maintained using established methods. The assay method is as previously described. Tests were run in triplicate in two independent runs to generate ten point, dose response curves to determine the half maximal effective concentration against the 3D7 and K1 P. falciparum strains for each drug. EC50 values were calculated with the robust investigation of screening experiments algorithm with a four parameter logistic equation. EC50 values of 1 uM were considered significant. GSK Tres Cantos used Dacomitinib a whole cell hypoxanthine radioisotope incorporation assay to determine per cent parasite inhibition at 48 hours and 96 hours. Plasmodium falciparum 3D7A strain was maintained as described previously.