PANK assay

PANK activity was monitored using a radioactive labeled PANK assay. The reaction mixtures contained 10 mM MgCl₂, 2.5 mM adenosine triphosphate, 5 μM ¹⁴C-labeled pantothenate, 100 mM tris-HCl (pH 7.4), the to-be-tested pantothenamide and enzyme (freshly prepared P. falciparum lysate), and recombinant protein in a total volume of 40 μl. Reactions were initiated after addition of enzyme and incubated at 37°C for 60 min. Reaction was terminated with 4 μl of a 10% acetic acid solution in 95% ethanol. Samples were loaded on diethylaminoethyl filter discs (GE Healthcare) and washed thoroughly in 1% acetic acid solution in 95% ethanol. Phosphorylated pantothenate will stay on the filter, whereas the unphosphorylated pantothenate will be washed away. After the discs were dried, they were transferred into scintillation vials containing 3 ml of ScintiSafe 30% cocktail (Fischer Scientific, Hampton, NH, USA). Radioactivity in each vial was counted using a Tri-Carb 2900TR Liquid Scintillation Analyzer (Packard BioScience, Boston, MA). Inhibition assays were performed in the presence of compound in a serial dilution range varying from 100 μM to 10 nM. IC₅₀ values were calculated with GraphPad Prism version 5.03.