Pantothenate kinase assay

The ability of the test compounds to inhibit P. falciparum pantothenate kinase (PfPanK) was assessed with a radiometric pantothenate phosphorylation assay as described previously [39]. In short, the phosphorylation of [¹⁴C] pantothenate by P. falciparum lysates was measured in the presence or absence of a single concentration (100 μM, two biological replicates in duplicate technical repeats) of each test compound. Additionally, the activity of STK 668036 on PfPanK and two additional parasite kinases, namely hexokinase (PfHK), and choline kinase (PfChK), was tested at 2, 10 and 100 μM compound concentration, in two independent experiments. P. falciparum lysate was prepared from trophozoite stage parasites isolated from their host RBC by treatment with 0.05% (w/v) saponin, and washed (three times) in HEPES-buffered saline (125 mM NaCl, 5 mM KCl, 20 mM D-glucose, 25 mM HEPES, 1 mM MgCl₂, pH 7.1). Lysates were prepared in 10 mM potassium phosphate, pH 7.4, the concentration of cell lysates was determined from cell counts with an improved Neubauer hemocytometer, and aliquots of lysate were stored at -20 °C.[¹⁴C] pantothenate, [¹⁴C] 2-deoxyglucose and [¹⁴C] choline phosphorylation were assayed using Somogyi reagent as described earlier [39].