A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts

Curtis A. Thorne, Bonnie Lafleur, Michelle Lewis, Alison J. Hanson, Kristin K. Jernigan, David C. Weaver, Kari A. Huppert, Tony W. Chen, Chonlarat Wichaidit, Christopher S. Cselenyi, Emilios Tahinci, Kelly C. Meyers, Emily Waskow, Darren Orton, Adrian Salic, Laura A. Lee, David J. Robbins, Stacey S. Huppert, Ethan Lee

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

Original languageEnglish (US)
Pages (from-to)995-1006
Number of pages12
JournalJournal of Biomolecular Screening
Volume16
Issue number9
DOIs
StatePublished - Oct 1 2011

Keywords

  • β-catenin
  • Axin
  • flavonoids
  • LRP6
  • Wnt signaling
  • Xenopus extracts

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Pharmacology
  • Biochemistry
  • Molecular Medicine
  • Biotechnology

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  • Cite this

    Thorne, C. A., Lafleur, B., Lewis, M., Hanson, A. J., Jernigan, K. K., Weaver, D. C., Huppert, K. A., Chen, T. W., Wichaidit, C., Cselenyi, C. S., Tahinci, E., Meyers, K. C., Waskow, E., Orton, D., Salic, A., Lee, L. A., Robbins, D. J., Huppert, S. S., & Lee, E. (2011). A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts. Journal of Biomolecular Screening, 16(9), 995-1006. https://doi.org/10.1177/1087057111416657