Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.
We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa.
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: benetode@hotmail.com.
Benetode
B
Konziase
D000345:Affinity Labels / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D000981:Antiprotozoal Agents / Q000737:chemistry* / Q000494:pharmacology; D037621:Artemisinins / Q000737:chemistry* / Q000494:pharmacology; D019921:Biotinylation; D015153:Blotting, Western; D006801:Humans; D058990:Molecular Targeted Therapy; D015800:Protozoan Proteins / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000378:metabolism*; D014353:Trypanosomiasis, African / Q000188:drug therapy / Q000469:parasitology
D000345:Affinity Labels; D000981:Antiprotozoal Agents; D037621:Artemisinins; D015800:Protozoan Proteins; C031327:artemisinin
Affinity labeling; Artemisinin; Biotinylated probes; Synthesis; Target proteins; Trypanosoma