Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei Stefanie Zimmermann a,b , Mouhssin Oufir a , Alejandro Leroux c , R. Luise Krauth-Siegel c , Katja Becker d , Marcel Kaiser b , Reto Brun b , Matthias Hamburger a , Michael Adams a,⇑ a Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland b Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland c Center of Biochemistry, Heidelberg University, Heidelberg, Germany d Research Center for Biochemistry, Justus-Liebig University of Giessen, Giessen, Germany article info Article history: Received 14 June 2013 Revised 16 August 2013 Accepted 23 August 2013 Available online 5 September 2013 Keywords: Sesquiterpene lactone Trypanosoma brucei Trypanothione Drug target HPLC–MS/MS abstract 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 a,b- 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 metab- olism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and orni- thine 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 com- ponents of the trypanosomal thiol metabolism. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Human African Trypanosomiasis (HAT) is a deadly parasitic dis- ease, which is caused by Trypanosoma brucei sub-species, and spread by the bite of the tsetse fly (Glossina spp.). As there is no vaccination chemotherapy remains the principal control of HAT. Severe side effects, treatment failures and complex administration schemes urgently demand for safer orally administrable drugs. 1 Several pathways in trypanosomes may provide suitable targets for new drugs including ergosterol- and purin biosynthesis, various kinases, farnesyl transferase, proteases, pyrimidine biosynthesis, compartmentalized glycolysis, and finally trypanothione-based re- dox metabolism (Fig. 6), with which a reducing intracellular milieu is maintained. 2–4 Because the trypanothione redox system is un- ique to trypanosomatids it represents several promising drug tar- gets such as trypanothione synthetase (TryS), trypanothione reductase (TR), spermidine synthase (SpS), and ornithine decarbox- ylase (ODC), or by direct interaction with glutathione (GSH) and trypanothione (T(SH) 2 )(Fig. 6). 5 We recently reported that cynaropicrin (CYN), a sesquiterpene lactone (STL) found in artichokes (Cynara scolymus L.) and some species of cornflowers (Centaurea spp.), inhibits the proliferation of T. b. rhodesiense in the acute mouse model. 6 CYN is the so far only plant compound demonstrated to have in vivo anti T. b. rhodesiense activity. Numerous other STLs have, however, shown antitrypanosomal effects in vitro. 7 Schmidt et al. supplied two excellent reviews of antiprotozoal in vitro effects of 883 plant derived natural products including 83 STLs. 8,9 The authors showed in a QSAR study of 40 STLs, that the antitrypanos- omal activity was linked to the presence of the a-methylene-c-lac- 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.08.052 Abbreviations: CYN, cynaropicrin; DMFO, eflornithine; DTT, dithiothreitol; FA, formic acid; GSH, glutathione; HAT, Human African Trypanosomiasis; IS, internal standard; LLOD, lower limit of detection; LLOQ, lower limit of quantification; ODC, ornithine decarboxylase; SAR, structure activity relationship; SpS, spermidine synthase; SS, stock solution; STL, sesquiterpene lactone; TR, trypanothione reduc- tase; TryS, trypanothione synthetase; T(SH) 2 , trypanothione. ⇑ Corresponding author. Tel.: +41 61 267 15 64; fax: +41 61 267 14 74. E-mail address: michael.adams@unibas.ch (M. Adams). Bioorganic & Medicinal Chemistry 21 (2013) 7202–7209 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc