title: A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts.
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.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8240, USA.
authors.forename: Curtis A
authors.initials: CA
authors.lastname: Thorne
authors.forename: Bonnie
authors.initials: B
authors.lastname: Lafleur
authors.forename: Michelle
authors.initials: M
authors.lastname: Lewis
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Hanson
authors.forename: Kristin K
authors.initials: KK
authors.lastname: Jernigan
authors.forename: David C
authors.initials: DC
authors.lastname: Weaver
authors.forename: Kari A
authors.initials: KA
authors.lastname: Huppert
authors.forename: Tony W
authors.initials: TW
authors.lastname: Chen
authors.forename: Chonlarat
authors.initials: C
authors.lastname: Wichaidit
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Cselenyi
authors.forename: Emilios
authors.initials: E
authors.lastname: Tahinci
authors.forename: Kelly C
authors.initials: KC
authors.lastname: Meyers
authors.forename: Emily
authors.initials: E
authors.lastname: Waskow
authors.forename: Darren
authors.initials: D
authors.lastname: Orton
authors.forename: Adrian
authors.initials: A
authors.lastname: Salic
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lee
authors.forename: David J
authors.initials: DJ
authors.lastname: Robbins
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Huppert
authors.forename: Ethan
authors.initials: E
authors.lastname: Lee
mesh_terms: D000818:Animals; D060466:Axin Protein / Q000378:metabolism; D057075:Enzyme Assays; D047309:Flavones / Q000494:pharmacology; D057809:HEK293 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D014982:Xenopus laevis / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D060466:Axin Protein; D047309:Flavones; D054852:Small Molecule Libraries; D051176:beta Catenin; D008156:Luciferases