Figure 4. Imidazopyrazines and quinoxalines bind the ATP-binding site of PI4K and alter PI4P intracellular distribution.

a, In silico docking of a homology model of the PfPI4K catalytic domain showing KAI407 (green) and BQR695 (orange) binding to the ATP-binding pocket of PfPI4K (cartoon representation; grey). Resistance SNVs (yellow) are labeled. b, Zoomed view of the ATP-binding pocket rotated 90° along the y-axis, showing the predicted hydrogen bond (green dash) between the imidazole nitrogen in KAI407 and the amide bond from the V1357 backbone (grey sticks). c, d, Recombinant full-length P. vivax PI4K (PvPI4K) activity in the presence of (c) KDU691 or (d) BQR695 monitored across a range of ATP concentrations (1.25–40 μM). Data shown as means±s.d. (n=9). e, Recombinant PvPI4K mutants S1286L, Y1322F or H1450Y (equivalent to PfPI4K-S1320L, -Y1356F or -H1484Y respectively) were assayed against imidazopyrazine and quinoxaline compounds. Fold-change in IC₅₀ value is shown relative to wild type enzyme (means±s.d.; n=9). Statistical significance was determined by the Mann–Whitney U test: *P<0.05; **P<0.01; ***P<0.001 (also for panel h). f, Illustration of the PI4P-dependent localization of GFP-PH^Osh2. g, In vivo distribution of PI4P was detected in parasites expressing GFP-PH^Osh2. In untreated parasites, GFP-PH^Osh2 localized to intracellular foci (blue arrow) and the plasma membrane (white arrow). Treatment with 500 nM KAI407 or BQR695 for 4 hr depleted the intracellular pool and redistributed the probe to the plasma membrane. Representative images from a single experimental replicate are shown (n=3). Nuclei were stained with Hoechst 33342 (blue); DIC, differential interference contrast. Scale bar, 5 μm. h, Quantification of intracellular GFP-PH^Osh2-labeled foci after drug treatment (means±s.d.; n=3).