Antimicrob Agents Chemother. 2014 Feb 18. [Epub ahead of print]
Expression of the essential kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs
We have previously shown that genetic disruption of Toxoplasma gondii calcium dependent protein kinase 3 (TgCDPK3) affects calcium ionophore induced egress. We examined whether PfCDPK1, the closest homolog of TgCDPK3 in the malaria parasite Plasmodium 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 PfCDPK1's localization to the plasma membrane and its kinase activity. Interestingly, Toxoplasma expressing PfCDPK1 become 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 kinase activity of recombinant PfCDPK1, for their ability to inhibit ionophore induced Egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we find that six drugs affected this process selectively in PfCDPK1 expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivity of Dasatinib and PLX-4720 is 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.
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