Heterologous Expression, Purification, and Functional Analysis of the Plasmodium falciparum Phosphatidylinositol 4-Kinase IIIβ

Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative III beta isoform (PfPI4KIII beta)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIII beta. We therefore codon optimized the pf pi4kIII beta gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIII beta protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIII beta activity is most consistent with the class III beta designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIII beta titrated enzyme inhibition for these candidates versus purified PfPI4KIII beta and PfVps34. We also analyzed the activity versus purified PfPI4KIII beta mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIII beta inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIII beta. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.