Characterization of 2,4-Dianilinopyrimidines Against Five P. falciparum Kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB.

Plasmodium kinases are increasingly recognized as potential novel antiplasmodial targets for the treatment of malaria, but only a small subset of these kinases have had structure-activity relationship (SAR) campaigns reported. Herein we report the discovery of CZC-54252 (1) as an inhibitor of five P. falciparum kinases PfARK1, PfARK3, PfNEK3, PfPK9, and PfPKB. 39 analogues were evaluated against all five kinases to establish SAR at three regions of the kinase active site. Nanomolar inhibitors of each kinase were discovered. We identified common and divergent SAR trends across all five kinases, highlighting substituents in each region that improve potency and selectivity for each kinase. Potent analogues were evaluated against the P. falciparum blood stage. Eight submicromolar inhibitors were discovered, of which 37 demonstrated potent antiplasmodial activity (EC50 = 0.16 µM). Our results provide an understanding of features needed to inhibit each individual kinase and lay groundwork for future optimization efforts toward novel antimalarials.

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 demonstrates the necessity to develop alternative antimalarials with novel mechanisms of action. We report the discovery of Ki8751 as an inhibitor of essential kinase PfPK6. 79 derivatives were designed, synthesized and evaluated for PfPK6 inhibition and antiplasmodial activity. Using group efficiency analyses, we established the importance of key groups on the scaffold consistent with a type II inhibitor pharmacophore. We highlight modifications on the tail group that contribute to antiplasmodial activity, cumulating in the discovery of compound 67, a PfPK6 inhibitor (IC50 = 13 nM) active against the P. falciparum blood stage (EC50 = 160 nM), and compound 79, a 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).

Identification of Novel 2,4,5-Trisubstituted Pyrimidines as Potent Dual Inhibitors of Plasmodial PfGSK3/PfPK6 with Activity against Blood Stage Parasites In Vitro.

Essential plasmodial kinases PfGSK3 and PfPK6 are considered novel drug targets to combat rising resistance to traditional antimalarial therapy. Herein, we report the discovery of IKK16 as a dual PfGSK3/PfPK6 inhibitor active against blood stage Pf3D7 parasites. To establish structure-activity relationships for PfPK6 and PfGSK3, 52 analogues were synthesized and assessed for the inhibition of PfGSK3 and PfPK6, with potent inhibitors further assessed for activity against blood and liver stage parasites. This culminated in the discovery of dual PfGSK3/PfPK6 inhibitors 23d (PfGSK3/PfPK6 IC50 = 172/11 nM) and 23e (PfGSK3/PfPK6 IC50 = 97/8 nM) with antiplasmodial activity (23d Pf3D7 EC50 = 552 ± 37 nM and 23e Pf3D7 EC50 = 1400 ± 13 nM). However, both compounds exhibited significant promiscuity when tested in a panel of human kinase targets. Our results demonstrate that dual PfPK6/PfGSK3 inhibitors with antiplasmodial activity can be identified and can set the stage for further optimization efforts.