A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

Article (PDF Available)inJournal of Biomolecular Screening 16(9):995-1006 · August 2011with42 Reads
DOI: 10.1177/1087057111416657 · Source: PubMed
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.


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Available from: Stacey S Huppert
    • "Thus, as for the Wnt pathway, a screening strategy using Xenopus egg extract represents an attractive approach to identify small molecule modulators of the Hedgehog and Notch pathways. Evidence for the feasibility of such an approach comes from our recent studies showing that the transcriptional mediators of the Hedgehog (Gli1) and Notch (NICD) pathways readily undergo robust degradation in Xenopus egg extract [81]. As with any biochemical approach, an obvious drawback to using the Xenopus extract system to screen for inhibitors of Wnt, Hedgehog, and Notch pathways is that one cannot screen for modulators of cellular morphology. "
    [Show abstract] [Hide abstract] ABSTRACT: Extract prepared from Xenopus eggs represents a cell-free system that has been shown to recapitulate a multitude of cellular processes, including cell cycle regulation, DNA replication/repair, and cytoskeletal dynamics. In addition, this system has been used to successfully reconstitute the Wnt pathway. Xenopus egg extract, which can be biochemically manipulated, offers an ideal medium in which small molecule screening can be performed in near native milieu. Thus, the use of Xenopus egg extract for small molecule screening represents an ideal bridge between targeted and phenotypic screening approaches. This review focuses on the use of this system for small molecules modulators of major signal transduction pathways (Notch, Hedgehog, and Wnt) that are critical for the development of the early Xenopus embryo. We describe the properties of Xenopus egg extract and our own high throughput screen for small molecules that modulate the Wnt pathway using this cell-free system. We propose that Xenopus egg extract could similarly be adapted for screening for modulators of the Notch and Hedgehog pathways.
    Full-text · Article · Mar 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Maternal Wnt/β-Catenin signaling is essential to establish dorsal-specific gene expression required for axial patterning in Xenopus. Deregulation of this pathway causes axis phenotypes in frog embryos. In adult life, mutations in the Wnt pathway components are associated with many diseases, such as polyposis coli; osteoporosis-pseudoglioma syndrome (OPPG); skeletal dysplasia; neural tube defects, cancer and many others. Thus, a better understanding of Wnt/β-catenin signaling will have great and significant impact on Biology and Medicine. In this aspect, natural compounds are potential targets as novel molecules that could modulate the Wnt pathway. For instance, flavonoids are a large group of natural compounds found in plants that modulate important cellular and molecular mechanisms related to diseases, but the specific in vivo mechanisms of action of most flavonoids remain unknown. In this way, Xenopus embryos may provide an efficient model, since it is frequently used to test and identify the role of molecules that affect Wnt/β-catenin signaling. Here, we describe a combination of approaches to outline and characterize the role of two flavonoids, quercetin and rutin, on Wnt/β-catenin signaling, using Xenopus embryos as an experimental model. Our data support that quercetin is potential in vivo modulator of canonical Wnt signaling and that this effect might depend on the structure of this molecule, as we did not observe any effect with rutin treatment, a flavonol structurally-related to quercetin. This model is useful to analyze effects of quercetin and other flavonoids in vivo and to provide further understanding of how natural compounds can modulate signaling pathways, using Xenopus embryos as a fast and efficient reading of in vivo effects of those compounds.
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  • [Show abstract] [Hide abstract] ABSTRACT: Cancer therapeutic development has historically driven Wnt drug discovery based on the biology of pathway mutations and because clinical trials can be rapidly progressed for patients with late-stage terminal disease. This chapter considers small-molecule and biological therapeutics that modulate the canonical Wnt signaling pathway upstream of β-catenin/TCF-dependent transcription. Initially, it is structured according to a linear order of “core” Wnt pathway components, Wnt Fzd/LRP Dvl–|Axin/APC/GSK3/CK1–|β-catenin TCF, and briefly discusses how and where novel therapeutics function (with a comprehensive list of Wnt “canonical pathway” therapeutics detailed in Table). Later, it uses the concept of a “Wnt network” to frame a discussion of Wnt inhibitor toxicity and the use of Wnt inhibitors in combinatorial therapy. The chapter also presents related recent reviews that discuss disease-specific opportunities, the targeting of noncanonical Wnt pathways, and the importance of target validation.
    Full-text · Chapter · Mar 2014 · Current topics in medicinal chemistry
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