PfCLK3 as a Therapeutic Antimalarial Drug Target

Artemisinin resistance, the current frontline antimalarial, is threatening to significantly increase the global incidence of malaria. Hence, novel targets for development of next generation antimalarials is urgently required. Here I focus on the essential malaria protein kinase PfCLK3, involved in RNA processing, to investigate if it is a suitable pharmacological target for malaria treatment. The molecule TCMDC-135051, identified from a screen at GlaxoSmithKline, was used as a probe inhibitor of PfCLK3. The evidence presented here shows that TCMDC-135051 had parasiticidal activity at multiple stages of P. falciparum growth at low nanomolar potency, with an IC50 of 180 nM in asexual ring stage parasites. TCMCD-135051 is also potent against asexual P. knowlesi and P. berghei parasites, meeting cross species requirements for new antimalarial agents. Against recombinant CLK3 kinases, TCMDC-135051 demonstrated a high potency, with an IC50 value of ~40 nM towards PfCLK3, PvCLK3 and PbCLK3 kinases. Additionally, a mode of action behaviour suggestive of non-ATP competitive inhibition was observed. For target validation, a mutant PfCLK3 parasite line (PfCLK3_G449P) reduced TCMDC-135051 potency by ~1.5 fold log units compared to wild type. Long-term exposure of Dd2 parasites to TCMDC-135051 showed two single point mutations on the PfCLK3 gene, indicating TCMDC-135051 selectivity towards PfCLK3. Using the parasite reduction rate to investigate the speed of action, TCMDC-135051 demonstrate activity levels similar to dihydroartemisinin, a standard antimalarial. Furthermore, inhibition of PfCLK3 resulted in impaired splicing in wild type 3D7 parasites compared to mutant PfCLK3_G449P, demonstrating the role of PfCLK3 in regulating RNA splicing. In conclusion, inhibition of PfCLK3 activity results in rapid killing of asexual P. falciparum parasites and other Plasmodium species at multiple stages through inhibition of RNA splicing. Therefore, the data presented here revealed PfCLK3 as a suitable target for treatment of symptomatic malaria and a potential transmissionblocking target.