Characterization of SP600126 for pharmacological inhibition of c-Jun N-terminal kinase activity in Schistosoma mansoni infected mice

Introduction: Schistosomiasis is a neglected tropical disease, affecting over 250 mio. people worldwide. Paired adult S. mansoni living in the mesenteric vein system produce about 300 eggs daily, eventually causing granulomatous liver-fibrosis. S. mansoni-infection permanently induces c-Jun, responsible for hepatocellular regeneration, proliferation and apoptosis. c-Jun is activated via phosphorylation by its specific kinase JNK. Prior to animal experiments, the inhibitory effect of the JNK-inhibitor SP600125 on S. mansoni SmJNK had to be characterized and compared to human hJNK.

Aims: The study intended to specify comparability and differences in the inhibitory potential of SP600125 on hJNK1 and SmJNK and whether this affects the vitality of adult S. mansoni.

Methods: The inhibitors binding to both organisms JNK was analysed in silico by molecular docking. Subsequently, a cellular thermal shift assay (CETSA) was performed to determine whether SP600125 binds to SmJNK. The binding affinity was further ascertained via an isothermal dose-response fingerprint (ITDRF) for intracellular SmJNK. A total of 30 mio. SmJNK-expressing Sf21 cells per treatment were used to establish the melting curve. Cells were cultured in presence of 10 mM SP600125 or DMSO (control) for 1 hour prior to the CESTA. To establish an ITDRF increasing concentrations of SP600125 were used and cells were heated at the TM of SmJNK.

Results: The reconstruction of the molecular structures and docking analysis suggest a slightly lower binding affinity between SmJNK and SP600125 compared to hJNK1. This can be explained by amino acid variations around the ATP-binding sites of the different JNK orthologues, where also SP600125 binds. In vitro treatment of adult S. mansoni with SP600125 caused no effects on motility, attachment and pairing ability.

Conclusion: The results suggest an inter-species variability in the binding of SP600125 regarding hJNK and SmJNK. Although we obtained evidence for binding of SP600125 to SmJNK, in vitro treatment of adult worms failed to affect worm fitness and viability. This may indicate a minor physiological role of JNK for adult S. mansoni or functional redundancy by other kinases compensating the inhibitory effect. Furthermore, we cannot exclude that the inhibitor may not have entered all relevant worm tissues under the in vitro conditions. As mouse cells show an almost identical JNK structure to human cells, the results listed are applicable to mouse models.

 

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Publication History

Article published online:
04 September 2025