Inhibition of PfCLK3 interferes with malaria parasite RNA splicing and explains the mechanism of action of a new class of antimalarial candidates

Emerging resistance to front-line antimalarials means there is an urgent need to discover new drugs with novel mechanisms of action that remain effective across multiple stages of the parasite life cycle. Previously, we reported a malaria parasite protein kinase, Pf CLK3, as a target offering a cure, prophylaxis, and transmission blocking. The homology between Pf CLK3 and human kinases suggested that the mechanism of parasiticidal activity of Pf CLK3 inhibitors is disruption of RNA processing. Here, we use RNA sequencing to reveal that selective Pf CLK3 inhibition extensively affects RNA splicing with 2,039 splice-junctions across 1,125 genes mis-spliced in treated wild-type parasites compared to controls. The function of mis-spliced transcripts showed that the affected genes were involved in numerous essential parasite processes associated with multiple life cycle stages and revealed transcripts and introns particularly susceptible to inhibition. Our study supports the role of Pf CLK3 as an important regulator of spliceosome activity and establishes the distinct mechanism of parasiticidal activity of Pf CLK3 inhibitors from current front-line treatments.