Fig. 6: PKIS library screen allows for the identification of several pan-FIKK kinases inhibitors that target at least one FIKK kinase in ATP-depleted iRBCs.
From: The fast-evolving FIKK kinase family of Plasmodium falciparum can be inhibited by a single compound
a, FIKK8 activity in the presence of increasing concentrations of staurosporine analogues (GW272220X, SB-219551, GW442389X, GW471214X, GW470969X and SB-505576). n = 6 technical replicates for each inhibitor. Shown is the mean ± s.e.m. b, A ranked plot showing the results of the PKIS library screen on recombinant FIKK8. A threshold of >75% inhibition was arbitrarily set and identified the 12 most potent PKIS compounds on recombinant FIKK8 kinase domain (n = 2). Each data point represents the mean percentage inhibition in both replicates. c, A SAR assay identifies closely related compounds with different behaviours towards recombinant FIKK8 kinase domain. A total of 333 compounds were identified from the three original PKIS chemical templates. The IC50 on recombinant FIKK8 kinase domain was measured in biological triplicate for each one of the compounds and are indicated here for the selected ones ±s.d. d, A heat map representing inhibition (%) of selected compounds on recombinant FIKK kinase domains (n = 3 biological replicates). e, Western blot showing adducin S726 phosphorylation in RBCs pretreated with 1,228 µM iodoacetamide and 2,046 µM inosine, infected with WT NF54 P. falciparum and treated with different concentrations of either GSK2236790B or GW779439X. The GAP50 antibody demonstrates equal loading. f, Western blot showing adducin S726 phosphorylation in RBCs pretreated with 1,228 µM iodoacetamide and 2,046 µM inosine, infected with FIKK1 conditional knockout (cKO) DMSO-treated P. falciparum and treated with different concentrations of either GSK2236790B or GW779439X. GAPDH antibody demonstrates equal loading. g, Immunofluorescence assays showing adducin S726 phosphorylation and protein export in ATP-depleted iRBC treated with different concentrations of either GSK2236790B or GW779439X. Protein export is investigated with anti-HA antibodies targeting the C-terminal HA-tag fused to FIKK1 kinase domain and with anti-SBP1 antibodies. DAPI (blue) is used as a nuclear staining. Scale bars, 5 µm. The immunofluorescence assays in e, f and g were performed at least three times with similar results.
