First isolated in the 1970s, artemisinin is an effective drug against malaria, but questions remain about how it works. With the help of chemical proteomics, researchers have now shown that artemisinin kills the malaria parasite Plasmodium falciparum by indiscriminately binding to proteins in many of the organism’s key biochemical pathways.

To identify the drug’s binding partners, a team led by Qingsong Lin, Kevin S. W. Tan, and Jigang Wang of National University of Singapore synthesized an alkyne-tagged artemisinin analog (Nat. Commun. 2015, DOI: 10.1038/ncomms10111). After incubating the probe with live malaria parasites and attaching biotin to its alkyne tag, the team fished out all the proteins to which artemisinin binds. The researchers did this by incubating the mixture with streptavidin-labeled beads that strongly attract biotin. They then identified the purified proteins with mass spectrometry.

The researchers were unsurprised to find that the probe bound to a whopping 124 different proteins. “The highly reactive and fast-acting nature of activated artemisinin suggests that it most likely has multiple targets,” Lin says. The fact that the drug has developed only low-level resistance after decades of extensive use bolsters this idea, too.

The drug’s promiscuity was first suggested by Steven R. Meshnick’s research team, which reported more than 20 years ago that radioisotope-labeled artemisinin binds to several parasite proteins (Antimicrob. Agents Chemother. 1994, DOI: 10.1128/aac.38.8.1854). Most of the proteins weren’t identified at the time, though, Wang explains.

In other experiments, the Singapore team also found that artemisinin needs heme, an iron-containing component of the red blood protein hemoglobin, to be activated. At different stages of the parasite life cycle, the heme comes from different sources. During an early stage, the heme comes from the parasite’s own biosynthetic pathway. At later stages in red blood cells, the heme comes from digested hemoglobin.

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“The central finding is consistent with previous lines of thought: Artemisinin has a fuse that is lit in the heme-rich parasite, where it then indiscriminately attacks like a bomb,” says Leila S. Ross, a postdoc studying malaria drug resistance in David A. Fidock’s lab at Columbia University. “Artemisinin kills by jamming up a large variety of cellular processes rather than a single pathway.”

But calling all of them “targets” of the drug would be a stretch, she says: “They seem to just be high-abundance proteins in the wrong place at the wrong time.”

Back when his team began studying artemisinin’s mechanism of action, Meshnick says, this idea was controversial, and technology at the time did not allow identification of the proteins. “Now, this elegant paper makes a convincing argument for heme-activated promiscuous protein modification,” says Meshnick, currently at the University of North Carolina, Chapel Hill. “This mechanism ties together the loose threads found in previous mechanisms.”