Discovery of Potent Plasmodium falciparum Protein Kinase 6 (PfPK6) Inhibitors with a Type II Inhibitor Pharmacophore

Malaria is a devastating disease that causes significant global morbidity and mortality. The rise of drug resistance against artemisinin-based combination therapy, the frontline treatment for malaria, demonstrates the necessity to develop alternative antimalarials with novel mechanisms of action. Inhibition of Plasmodium protein kinases presents an underexplored opportunity for drug development. PfPK6 has been identified as an essential kinase for P. falciparum asexual blood stage proliferation, but has not been subjected to medicinal chemistry campaigns for inhibitor development. In this work, we report the discovery of Ki8751 as a PfPK6 inhibitor (IC50 = 14 nM) determined using the KinaseSeeker assay, utilizing split-luciferase three-hybrid technology. A series of 79 1-phenyl-3-(4-(quinolin-4-yloxy)phenyl)urea derivatives of Ki8751 were designed, synthesized and evaluated for PfPK6 inhibition and antiplasmodial activity. Using group efficiency analyses, we established the importance of the key groups on the scaffold for inhibition of PfPK6 consistent with a type II inhibitor pharmacophore. We highlight modifications on the tail group that contribute to antiplasmodial activity. We report the discovery of compound 67, a potent PfPK6 inhibitor (IC50 = 13 nM) active against the P. falciparum blood stage (EC50 = 160 nM), and compound 79, an excellent PfPK6 inhibitor (IC50 < 5 nM) with dual-stage antiplasmodial activity against P. falciparum blood stage (EC50 = 39 nM) and against P. berghei liver stage (EC50 = 220 nM). These results lay the foundation for this chemotype to be further developed into novel antimalarials and into chemical probes targeting PfPK6, enabling further investigation into PfPK6 function.