Target identification and mechanism of action in chemical biology and drug discovery

Abstract

Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.

Figure 1. Mechanism-of-action and target identification in chemical genetics

(a) Target-based approaches (reverse chemical genetics) begin with target validation, in which a role is established for a protein in a pathway or disease, followed by a biochemical assay to find candidate small molecules; mechanism-of-action studies are still required to validate cellular activities of candidates and evaluate possible side effects. (b) Phenotype-based approaches (forward chemical genetics) begin with a phenotype in a model system and an assay for small molecules that can perturb this phenotype; candidate small molecules must then undergo target-identification and mechanism-of-action studies to determine the protein responsible for phenotypic change.

Figure 2. Illustration of stable isotope labeling and quantitative MS

Cells are labeled with either heavy- or light-isotope labels. One sample is exposed to bead-immobilized small molecules (SM) in the presence of soluble competitor compound and the other in the absence of competitor. Following mixing, washing and electrophoresis, samples are digested using trypsin and peptide fragments analyzed by quantitative MS. Ratios of heavy- and light-labeled peptides are used to determine specificity of interactions for the small molecule, potentially including both direct and indirect targets (for example, members of complexes including the direct target), but not to differentiate them.

Figure 3. Illustrations of yeast genomic methods for target-identification and mechanism-of-action studies

(a) A panel of viable single-gene deletions is tested for small-molecule (SM) sensitivity, indicating synthetic-lethal interactions between potential targets and the original deletion; mechanisms are interpreted by comparing interactions to double-knockout strains. (b) Different strains of diploid yeast are mated to form F1 recombinants, and meiotic progeny are subjected to small molecules; segregation frequencies allow mapping of small-molecule sensitivity to genetic loci. (c) A recessive small molecule–resistant mutant is transformed with a wild-type open reading frame library; transformants obtaining a wild-type copy of the mutant gene are selectively sensitive to small molecules, resulting in their depletion among pooled transformants, as quantified by microarray.

Figure 4. Illustrations of RNAi-based methods for target-identification and mechanism-of-action studies

(a) In one implementation, phenotypes from genome-wide RNAi are compared to those induced by a small molecule (SM) of interest; full or partial phenocopy of the small-molecule effect by RNAi provides evidence that the gene product is a small-molecule target. (b) When prior evidence suggests a particular target pathway, focused sets of RNA reagents can help to generate mechanistic hypotheses. In general, RNAi can enhance or suppress small-molecule effects, as in genetic epistasis analysis; in practice, more complex relationships among proteins than those illustrated may also exist.

Figure 5. Illustration of computational inference methods for target-identification and mechanism-of-action studies

In general, data sets that provide multidimensional readouts of small molecule (SM)-induced phenotypes can be used to provide connections between new small-molecule signatures and reference databases by similarity to ‘landmark’ compounds with known mechanisms of action.

Figure 6. Illustration of a conceptual workflow for integrated target-identification and mechanism-of-action studies

Small-molecule discovery often starts with phenotypic screening, and, depending on the expertise available to the researchers, target identification could proceed using any combination of direct biochemical methods, genetic or genomic methods or using computational inference methods. A key component for success is the integration of data from all available methods to produce the most reliable target and mechanistic hypotheses.