Artemisinin (ART)-based combination therapies are the current first-line treatment for Plasmodium falciparum malaria. Youyou Tu was awarded a share in The Nobel Prize for Medicine in 2015 for her discovery of artemisinin, yet the mechanism of action of this life saving drug remains largely unknown. Previous studies have revealed that ARTs do not target one specific protein or organelle, but rather bind promiscuously to hundreds of different parasite proteins throughout many compartments of the cell. We therefore hypothesised that ART-induced parasite death is due to widespread protein damage and disruption of protein homeostasis.
Here we show, using the novel cell-permeable thiol probe, tetraphenylethene maleimide (TPE-MI), that treatment of P. falciparum with the clinically relevant ART derivative dihydroartemisinin (DHA), causes an increase in the unfolded protein load of the cell, leading to activation of the parasite’s Unfolded Protein Response (UPR) pathway and resulting in arrest of protein translation. Using genetic knockdown, we identify Protein Kinase 4 (PK4), as the mediator of both the UPR and the response to DHA.
We further show that DHA prevents degradation of unfolded/misfolded protein by inhibiting activity of the proteasome, resulting in a dramatic accumulation of polyubiquitinated proteins. We find that co-treatment of parasites with DHA and chemical inhibitors of ubiquitination; prevents polyubiquitinated proteins from accumulating, reduces the level of ER stress and almost completely rescues parasites from DHA-induced killing. These findings highlight proteasome inhibition, accumulation of polyubiquitinated proteins and ER stress as critical events in ART-mediated parasite death and suggests that proteostatic stress is a key point of vulnerability that should be targeted in the development of new antimalarials.