Marc Kaethner (Bern / CH), Pascal Zumstein (Bern / CH), Matías Preza (Bern / CH), Philipp Grossenbacher (Bern / CH), Martin Lochner (Bern / CH), Stefan Schürch (Bern / CH), Clement Regnault (Glasgow / GB), Daniel Villalobos Ramírez (Würzburg / DE), Britta Lundström-Stadelmann (Bern / CH)
Abstract text
Introduction
Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode of the fox tapeworm Echinococcus multilocularis. Novel treatment options are urgently needed. In vitro experiments showed strong scavenging of threonine by E. multilocularis metacestodes. Metacestodes express a threonine dehydrogenase (EmTDH) that is potentially a future drug target against AE, as human TDH is non-functional.
Objectives
We aimed to investigate the importance of threonine uptake and metabolism for E. multilocularis and to study whether threonine-degrading pathways can be targeted for the development of new drugs against the parasite. Quinazoline carboxamids (QCs) are potent inhibitors of mouse TDH and we aimed to test them against E. multilocularis.
Material and methods
13C4 L-threonine and metabolites were traced in in vitro cultured metacestodes to give insights into the metabolization of threonine in E. multilocularis. In addition, we studied the effect of L-threonine on the growth of in vitro cultured metacestodes and on the vesicle formation of E. multilocularis primary cells. QCs were tested against E. multilocularis metacestodes via damage marker release assay and in enzymatic assays against EmTDH activity in crude extracts of metacestodes as well as recombinantly expressed EmTDH.
Results
Flux analysis with 13C4 L-threonine showed that E. multilocularis metacestodes take up threonine and metabolize it to glycine, which is indicative for a functional TDH pathway. Significantly increased growth was shown for threonine-treated metacestodes. Increased numbers of vesicles were formed upon treatment of primary cell cultures with threonine, whereas the threonine analogue 3-hydroxy norvaline reduced the number of vesicles. One QC inhibited EmTDH in an enzymatic assay using crude extract of metacestodes and it was toxic against E. multilocularis metacestodes. However, recEmTDH was not inhibited by any of the here tested QCs.
Conclusion
Flux analysis, growth assays and vesicle formation assays provided evidence that threonine metabolism is important for E. multilocularis and inhibiting this metabolism could provide new treatment options. Further investigation of the in vivo situation are needed. Novel QC derivatives will be designed for successful inhibition of EmTDH to be tested in the here established enzymatic assays for medium throughput screening of potential other inhibitors targeting the threonine metabolism of E. multilocularis.