title: Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs.
abstract: We have previously shown that genetic disruption of Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3) affects calcium ionophore-induced egress. We examined whether Plasmodium falciparum CDPK1 (PfCDPK1), the closest homolog of TgCDPK3 in the malaria parasite P. falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility, and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires the localization of PfCDPK1 to the plasma membrane and kinase activity. Interestingly, PfCDPK1-expressing Toxoplasma becomes more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit the kinase activity of recombinant PfCDPK1 for their abilities to inhibit ionophore-induced egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we found that six drugs affected this process selectively in PfCDPK1-expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivities of dasatinib and PLX-4720 are regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1-specific inhibitors that can be developed as antimalarials.
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Rajshekhar Y
authors.initials: RY
authors.lastname: Gaji
authors.forename: Lisa
authors.initials: L
authors.lastname: Checkley
authors.forename: Michael L
authors.initials: ML
authors.lastname: Reese
authors.forename: Michael T
authors.initials: MT
authors.lastname: Ferdig
authors.forename: Gustavo
authors.initials: G
authors.lastname: Arrizabalaga
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000069439:Dasatinib; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014122:Toxoplasma / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D007211:Indoles; C528407:PLX 4720; D015800:Protozoan Proteins; D011743:Pyrimidines; D013449:Sulfonamides; D013844:Thiazoles; D011494:Protein Kinases; C531238:calcium-dependent protein kinase-1, Plasmodium falciparum; D000069439:Dasatinib