Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei.
In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei-the causative agent of Human African Trypanosomiasis-by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)2), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)2 redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC-MS/MS analysis was established to quantify intra-cellular CYN, T(SH)2, GSH, as well as GS-CYN and T(S-CYN)2 adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)2 pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)2 redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)2 redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.
Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland; Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland.
Stefanie
S
Zimmermann
Mouhssin
M
Oufir
Alejandro
A
Leroux
R Luise
RL
Krauth-Siegel
Katja
K
Becker
Marcel
M
Kaiser
Reto
R
Brun
Matthias
M
Hamburger
Michael
M
Adams
D019732:Amide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007783:Lactones / Q000737:chemistry* / Q000494:pharmacology* / Q000627:therapeutic use; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009955:Ornithine Decarboxylase / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D010084:Oxidation-Reduction; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry* / Q000494:pharmacology* / Q000627:therapeutic use; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology
D007783:Lactones; D065108:Ornithine Decarboxylase Inhibitors; D015800:Protozoan Proteins; D012717:Sesquiterpenes; D013438:Sulfhydryl Compounds; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D009955:Ornithine Decarboxylase; D019732:Amide Synthases; C064407:trypanothione synthetase; C075687:cynaropicrin
CYN; DMFO; DTT; Drug target; FA; GSH; HAT; HPLC–MS/MS; Human African Trypanosomiasis; IS; LLOD; LLOQ; ODC; SAR; SS; STL; Sesquiterpene lactone; SpS; T(SH)(2); TR; TryS; Trypanosoma brucei; Trypanothione; cynaropicrin; dithiothreitol; eflornithine; formic acid; glutathione; internal standard; lower limit of detection; lower limit of quantification; ornithine decarboxylase; sesquiterpene lactone; spermidine synthase; stock solution; structure activity relationship; trypanothione; trypanothione reductase; trypanothione synthetase