@article{b11ebf95c5b04e41960554ec96753f16,
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.",
keywords = "Axin, LRP6, Wnt signaling, Xenopus extracts, flavonoids, β-catenin",
author = "Thorne, {Curtis A.} and Bonnie Lafleur and Michelle Lewis and Hanson, {Alison J.} and Jernigan, {Kristin K.} and Weaver, {David C.} and Huppert, {Kari A.} and Chen, {Tony W.} and Chonlarat Wichaidit and Cselenyi, {Christopher S.} and Emilios Tahinci and Meyers, {Kelly C.} and Emily Waskow and Darren Orton and Adrian Salic and Lee, {Laura A.} and Robbins, {David J.} and Huppert, {Stacey S.} and Ethan Lee",
note = "Funding Information: We thank members of the E. Lee and L. Lee lab for critical reading of the manuscript, Ashley Spann and Jessica Sweatt for assistance with tissue culture and experiments, and members of the Institute of Chemistry and Cell Biology–Longwood for assistance with screening. This work was supported by American Cancer Society Research Scholar grant RSG-05-126-01, National Cancer Institute grant GI SPORE P50 CA95103, National Institutes of Health grant 1 R01 GM081635-01 (E. Lee), American Heart Association Predoctoral Fellowship 0615279B, Molecular Endocrinology Training grant 5 T 32 DK007563 (C. A. Thorne), National Institute of General Medical Studies Medical-Scientist Training grant 5 T32 GM007347, Ruth L. Kirschstein National Research Service Award 4042800401 (A. J. Hanson), American Heart Association Predoctoral Fellowships 0615162B, and National Institutes of Health Cancer Biology Training grant T32 CA09592 (K. K. Jernigan). C. Wichiadit is supported in the Altschuler and Wu labs by Welch grant (I-1644) and Eureka grant (GM085442). E. Lee is a recipient of a Pew Scholarship in the Biomedical Sciences. ",
year = "2011",
month = oct,
doi = "10.1177/1087057111416657",
language = "English (US)",
volume = "16",
pages = "995--1006",
journal = "Journal of Biomolecular Screening",
issn = "1087-0571",
publisher = "SAGE Publications Inc.",
number = "9",
}