Chemical Screening Using Cell-Free Xenopus Egg Extract

Abstract

Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as Xenopus, zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. Xenopus laevis egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, Xenopus egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, Xenopus egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, Xenopus egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsChemical librarySelect a library based on the specific screen. Energy reaction mix (ER mix) <R> Reagents for assay detectionKits are commercially available and should be chosen based on the specific application. Light-based readouts (e.g., luminescence, fluorescence, etc.) are favored in chemical screening for their robust signals, ease in execution, quantifiability, and rapidity of setup. Other types of readout (e.g., immunoblotting or radioactive-based assays) require considerable time and effort to perform and are cumbersome when large libraries are used.