Chemical genetics: ligand-based discovery of gene function

Abstract

Chemical genetics is the study of gene-product function in a cellular or organismal context using exogenous ligands. In this approach, small molecules that bind directly to proteins are used to alter protein function, enabling a kinetic analysis of the in vivo consequences of these changes. Recent advances have strongly enhanced the power of exogenous ligands such that they can resemble genetic mutations in terms of their general applicability and target specificity. The growing sophistication of this approach raises the possibility of its application to any biological process.

(Adapted with permission from Nature Med. (REF. 52) © (1999) Macmillan Magazines Ltd)

Figure 1. Genetic and chemical-genetic approaches identify genes and proteins, respectively, that regulate biological processes

a | Forward genetics entails introducing random mutations into cells, screening mutant cells for a phenotype of interest and identifying mutated genes in affected cells. In the example shown, yeast cells are randomly mutated, cells showing a large-bud phenotype are selected, and genes mutated in these cells are identified. Reverse genetics entails introducing a mutation into a specific gene of interest and studying the phenotypic consequences of the mutation in a cellular or organismal context. In the example shown, a single mutated gene is introduced into yeast cells and a large-bud phenotype is observed. b | Forward chemical-genetics entails screening exogenous ligands in cells, selecting a ligand that induces a phenotype of interest, and identifying the protein target of this ligand. In the example shown, one compound that induces a large-bud phenotype is selected and the protein target of this ligand is subsequently identified. Reverse chemical-genetics entails overexpressing a protein of interest, screening for a ligand for the protein, and using the ligand to determine the phenotypic consequences of altering the function of this protein in a cellular context. In the example shown, a ligand for a specific protein is found to induce a large-bud phenotype.

Figure 2. Small molecules have a large range of structural complexity

Simple small molecules, such as a | methylene blue, b | crystal violet, c | aspirin and d | saccharin, lack STEREOCENTRES, whereas complex small molecules, such as e | morphine, f | FK506, g | cyclosporin and h | peptides, have one or more stereocentres.

Figure 3. High-throughput cell-based assays

These assays measure the effect of a collection of exogenous ligands on a specific biological process. a | To screen a large number of small molecules, a microtitre plate is prepared with the cells of interest. b | A small volume of each compound to be tested is added to a well of the microtitre plate. c–e | One of several possible methods is used to detect the effect of each compound on the cells. c | An antibody detects post-translational or biosynthetic changes within the cell. d | A reporter-gene assay detects changes in gene expression at a specific promoter. e | Morphological or other changes in the cells are visualised by microscopy. In each case, those reagents that cause the desired cellular change are selected for further study (shown as the red well in b).