Chemical Complementation: A Genetic Selection System in Yeast for Drug Discovery, Protein Engineering, and for Deciphering and Assembling Biosynthetic Pathways

Abstract

Chemical complementation is a general system for detecting protein-small molecule interactions, and linking that interaction to genetic selection. In this chemical complementation system, the interaction of a nuclear receptor and a ligand is essential for yeast survival. In first generation chemical complementation, a two-component assay was developed where the Gal4 DNA-binding domain is fused to the ligand binding domains of nuclear receptors, and expressed in the strain S.cerevisiae PJ69-4A. The Gal4 DNA binding domain binds to a Gal4 response element controlling transcription of a selective marker, and the nuclear receptor ligand-binding domain binds its ligand. This system was developed using the retinoid X receptor, the pregnane X receptor, and the liver X receptor and their ligands 9-cis retinoic acid, paclitaxel, and oxysterols, respectively. Yeast survive on selective plates only in the presence of both components: a nuclear receptor and the corresponding ligand. Growth was observed at the highest concentration of ligand (10-5 M) and, compared to Gal4-activated growth, the growth density was less and growth time was more. The second generation chemical complementation system is a three-component system comprising a nuclear receptor ligand-binding domain fused to the Gal4 DNA binding domain, the ligand, and a nuclear receptor coactivator fused to the yeast Gal4 activation domain. This system was developed using the retinoid X receptor and has been extended to several other nuclear receptors. The sensitivity of chemical complementation is increased 1000-fold, and growth time and density are equivalent to Gal4-activated growth. An assay was developed to provide a quantitative high-throughput assay for evaluating nuclear receptor- ligand interactions, and measuring EC50 values for the ligand-receptor pairs. Chemical complementation can be used in a variety of applications, such as drug discovery for nuclear receptor-based disease, providing a high-throughput assay for the discovery of potential nuclear receptor agonists, and with the use of the negative chemical complementation system, the discovery of nuclear receptor antagonists. Chemical complementation is used for protein engineering, specifically engineering receptors to bind and activate in response to other ligands. Chemical complementation is also used for deciphering and assembling biosynthetic pathways.