Chemical proteomics approach reveals the direct targets and the heme-dependent activation mechanism of artemisinin in Plasmodium falciparum using an artemisinin-based activity probe

Abstract

Artemisinin and its analogues are currently the most effective anti-malarial drugs. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources. Once activated, artemisinins attack macromolecules through alkylation and propagate a series of damages, leading to parasite death. Even though several parasite proteins have been reported as artemisinin targets, the exact mechanism of action (MOA) of artemisinin is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted for. Recently, we have developed an unbiased chemical proteomics approach to directly probe the MOA of artemisinin in P. falciparum. We synthesized an artemisinin analogue with an alkyne tag, which can be coupled with biotin through click chemistry. This enabled selective purification and identification of 124 protein targets of artemisinin. Many of these targets are critical for the parasite survival. In vitro assays confirmed the specific artemisinin binding and inhibition of selected targets. We thus postulated that artemisinin kills the parasite through disrupting its biochemical landscape. In addition, we showed that artemisinin activation requires heme, rather than free ferrous iron, by monitoring the extent of protein binding using a fluorescent dye coupled with the alkyne-tagged artemisinin. The extremely high level of heme released from the hemoglobin digestion by the parasite makes artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages) compared to parasites at early ring stage, which have low level of heme, mainly derived from endogenous synthesis. Such a unique activation mechanism also confers artemisinin with extremely high specificity against the parasites, while the healthy red blood cells are unaffected. Our results provide a sound explanation of the MOA of artemisinin and its specificity against malaria parasites, which may benefit the optimization of treatment strategies and the battle against the emerging drug resistance.

Keywords: activation; activity-based probe; artemisinin; artemisinin resistance; chemical proteomics; heme; malaria; mechanism of action; targets.

Figure 1. FIGURE 1: Artemisinin, activated by heme, effectively kills the malaria parasites by covalently targeting over 120 targets like a bomb.

Artemisinin (chemical structure shown) can effectively kill the malaria parasites within the red blood cells (pictured). Wang et al. identified over 120 covalent protein targets of artemisinin. Many of them play important roles in different biological processes critical for the parasite survival. They also proved that artemisinin activation relies on heme, either biosynthesized by ring-stage parasites, or derived from hemoglobin digestion at later parasite stages.