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.

Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the Ribosome

“…33,34 Briefly, varying concentrations of the compounds of interest were allowed to incubate in a solution of rabbit reticulocyte or E. coli extract and all amino acids for 20 min at room temperature. Following brief centrifugation, 0.95 μL luciferase control template (Promega) was added to each tube.…”

Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel

“…Both assays utilize a luciferase based reporter, which measures the amount of luciferase protein produced, to indirectly quantify ribosome function. 37,38 We determined the IC 50 s for all the target compounds in addition to that of azithromycin which we used as a positive (Table 1). …”

Small-Molecule High-Throughput Screening Utilizing Xenopus Egg Extract

“…The method of Xenopus egg extract preparation described within this chapter is optimized for analyzing protein turnover of β-catenin, the key effector protein of the Wnt signaling pathway; also, we found that it supports the degradation of another Wnt component, Axin, as well as other signaling pathway proteins that are known to rapidly turn over [5, 37, 38]. The usefulness of Xenopus egg extract for studying key aspects of cytoplasmic Wnt pathway regulation is supported by multiple studies that identify important regulatory proteins/steps that contribute to β-catenin degradation [2, 3, 5, 38–44]. Significantly, the preparation of Xenopus egg extract described herein was successfully used to screen and identify small molecules that stimulate β-catenin turnover and inhibit Wnt signaling [2, 3].…”

“…In addition, Xenopus egg extract contains all of the eukaryotic cellular machinery and complex signaling pathways required for the early development of an organism. Finally, large amounts of homogenous Xenopus egg extract can be prepared at one time, an important consideration for large-scale screens and reproducibility [1–3]…”

“…The usefulness of Xenopus egg extract for studying key aspects of cytoplasmic Wnt pathway regulation is supported by multiple studies that identify important regulatory proteins/steps that contribute to β-catenin degradation [2, 3, 5, 38–44]. Significantly, the preparation of Xenopus egg extract described herein was successfully used to screen and identify small molecules that stimulate β-catenin turnover and inhibit Wnt signaling [2, 3]. …”