| Chemical complementation is a general system for detecting protein-small molecule interactions, and linking that interaction to genetic selection in yeast. Chemical complementation provides a general system for detecting a small molecule for a protein, such as a receptor or an enzyme. Here, chemical complementation is used to identify the interaction of a small molecule ligand with a nuclear receptor. 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 S. cerevisiae strain 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 (RXR), the pregnane X receptor (PXR), and the liver X receptor (LXR) and their ligands, 9-cis retinoic acid, paclitaxel, and oxysterols respectively. Yeast survive on selective plates only in the presence of two components: a nuclear receptor and the corresponding ligand. Growth was observed at the highest concentration of ligand (10-5 M) and the growth density was less than Gal4-activated growth. The growth time was also longer than Gal4-activated growth.; The 2nd generation chemical complementation system is a three-component system comprising a nuclear receptor ligand-binding domain fused to the Gal4 DNA binding domain, and a nuclear receptor coactivator fused to the yeast Gal4 activation domain. After the ligand binds to the nuclear receptor ligand-binding domain, a conformational change recruits the nuclear receptor coactivator fusion protein, inducing transcription via RNA polymerase. This system has been developed using the retinoid X receptor and 9-cis retinoic acid and has been extended to and tested with a 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 of chemical complementation was developed to provide a quantitative high-throughput assay for evaluating nuclear receptor-ligand interactions, and measuring EC 50 values for the ligand-receptor pairs. A correlation between the functions of nuclear receptors in yeast and mammalian cell assays is observed, therefore, chemical complementation an attractive system for developing and evaluating nuclear receptor-ligand interactions.; Chemical complementation can be extended to a variety of applications. The first application is for drug discovery, because nuclear receptors are implicated in a number of diseases, ranging from metabolic disorders to a variety of cancers, and are currently targets for ∼10% of commonly prescribed drugs and potentially many more. This system provides a high-throughput assay for the discovery of potential nuclear receptor ligands, such as agonists, and with the use of negative chemical complementation, the discovery of nuclear receptor antagonists. Drug discovery assays may be extended to enzyme targets by engineering receptors that activate transcription in response to the small molecule product of the enzyme-catalyzed reaction. In protein engineering applications, chemical complementation offers a general method of engineering receptors that activate transcription in response to arbitrary small molecules. This system uses the power of genetic selection to analyze libraries of protein variants in a more time and effort efficient method, allowing the survival of the variants with the desired functions. The discovery of these engineered protein-ligand pairs can serve as candidates for small molecule based gene regulation, with applications in gene therapy. Other applications of engineered receptors extend to biotechnological applications, such as biosensors, and for engineering enzymes with enhanced or novel functions. Finally, with the discovery of more and more nat... |
