In vitro activity: BQR-695 (previously known as BQR695 NVP-BQR695) is a potent and selective PI4KIIIβ inhibitor with IC50 values of 80 and 3.5 nM for human PI4KIIIβ and Plasmodium variant of PI4KIIIβ, respectively. The crystal structure of PI4KIIIβ in complex with the potent anti‐malarial compound BQR695 in complex with GDP loaded Rab11 has been solved at a resolution of 3.2 A. BQR-695 makes two putative hydrogen bonds with PI4KIIIβ, one between the nitrogen of the central quinoxaline and the amide hydrogen of V598, and one between the hydrogen on the amino group off the central quinoxaline with the carbonyl of A601.

Kinase Assay: One hundred nanometer extruded PI vesicles were made with soybean phosphatidylinositol (Sigma) in lipid buffer [20 mM HEPES pH 7.5 (RT), 100 mM KCl, 0.5 mM EDTA] using the Avanti lipid mini‐extruder. Lipid kinase assays were carried out using the Transcreener(R) ADP2 FI Assay (BellBrook Labs) following the published protocol as previously described; 4 μL Reactions ran at 21°C for 30 min in a buffer containing 30 mM Hepes pH 7.5 (RT), 100 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 0.4% v/v Triton‐X, 1 mM TCEP, 0.5 mg/mL PI vesicles and 10 μM ATP. Both full length human PI4KIIIB and the truncated PI4KIIIB crystal construct with the c‐ erm were run at 200 nM. Fluorescence intensity was measured using a Spectramax M5 plate reader with λex = 590 nm and λem = 620 nm (20‐ m bandwidth).

Cell Assay: A clonal population of P. falciparum Dd2 parasites is used to initiate two or three independent parasite cultures under the initial selection pressure of 12 nM KAI407, 1 nM KAI715 or 40 nM BQR695. Stepwise drug evolution continues until the final concentration is at least 3-fold higher than the initial concentration (typically 80 to 120 days). For each of the ten resistant strains, copy number variations (CNVs) and single nucleotide variations (SNVs) are detected using a whole-genome tiling array and analyzed with PfGenominator. The susceptibility of each resistant strain to KAI407, KAI715, KDU691 and BQR695 is determined by the 72-hr SYBR Green cell proliferation assay with four independent experiments assayed in duplicate.

BQR-695 (previously known as BQR695 or NVP-BQR695) is a potent and selective PI4KIIIβ inhibitor with antimalarial activity. It inhibits human PI4KIIIβ and Plasmodium variant of PI4KIIIβ with IC50 values of 80 and 3.5 nM, respectively. The crystal structure of PI4KIIIβ in complex with the potent anti‐malarial compound BQR695 in complex with GDP loaded Rab11 has been solved at a resolution of 3.2 A. BQR-695 makes two putative hydrogen bonds with PI4KIIIβ, one between the nitrogen of the central quinoxaline and the amide hydrogen of V598, and one between the hydrogen on the amino group off the central quinoxaline with the carbonyl of A601.