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

doi:10.1177/1087057111416657

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

Journal article

Peer reviewed

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, …

Journal of biomolecular screening, Vol.16(9), pp.995-1006

10/01/2011

DOI: 10.1177/1087057111416657

PMCID: PMC3694444

PMID: 21859680

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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.

Animals

Axin Protein - metabolism

beta Catenin - metabolism

Enzyme Assays

Flavones - pharmacology

HEK293 Cells

HeLa Cells

High-Throughput Screening Assays

Humans

Luciferases - metabolism

Reproducibility of Results

Small Molecule Libraries

Wnt Signaling Pathway - drug effects

Xenopus laevis - metabolism

Details

Title: Subtitle: A biochemical screen for identification of small-molecule regulators of the Wnt pathway using Xenopus egg extracts
Creators: Curtis A Thorne - Vanderbilt University Medical Center
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
Resource Type: Journal article
Publication Details: Journal of biomolecular screening, Vol.16(9), pp.995-1006
DOI: 10.1177/1087057111416657
PMID: 21859680
PMCID: PMC3694444
NLM abbreviation: J Biomol Screen
ISSN: 1087-0571
eISSN: 1552-454X
Grant note: P50 CA095103 / NCI NIH HHS R01 GM081635 / NIGMS NIH HHS GM085442 / NIGMS NIH HHS T32 DK007563 / NIDDK NIH HHS T32 CA009592 / NCI NIH HHS T32 CA09592 / NCI NIH HHS T32 GM007347 / NIGMS NIH HHS R01 GM085442 / NIGMS NIH HHS 5 T 32 DK007563 / NIDDK NIH HHS 1 R01 GM081635-01 / NIGMS NIH HHS
Language: English
Date published: 10/01/2011
Academic Unit: Psychiatry
Record Identifier: 9984823120202771

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