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Colorectal cancer remains the second leading cause of cancer-related mortalities worldwide. While artemisinin (ART), a key active compound from the traditional Chinese medicinal herb Artemisia annua , has been recognized for its antiproliferative activity against colon cancer cells, its underlying molecular underpinnings remain elusive. Whereas promiscuity of heme-dependent alkylating of macromolecules, mainly proteins, has been seen pivotal as a universal and primary mode of action of ART in cancer cells, accumulating evidence suggests the existence of unique targets and mechanisms of actions contingent on cell or tissue specificities. Here, we employed photoaffinity probes to identify the specific targets responsible for ART's anti-colon cancer actions. Upon validation, microsomal prostaglandins synthase-2 emerged as a specific and reversible target of ART in HCT116 colorectal cancer cells, whose inhibition resulted in reduced cellular prostaglandin E 2 biosynthesis and cell growth. Our discovery opens new opportunities for pharmacological treatment of colon cancer.

Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin-protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the "capture-and-release" strategy affords consolidated advantages previously unavailable in artemisinin-protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin.

The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets.