By monitoring the interaction between a chemical probe and its protein target in a model system, a researcher gains confidence that the resulting phenotypic changes can be attributed to the probe’s activity against that protein. Assays that allow a researcher to detect and ideally to quantify the protein-chemical probe binding interaction are referred to as target-engagement assays and are considered an essential part of the evidence required to properly validate a probe for use in a biological system.

For programs that use a protein target-based approach to generate a chemical probe, meaning they start with the protein of interest and try to find a small molecule that modulates its function, scientists typically develop an assay using a recombinant purified version of the protein in vitro that allows them to identify small molecules that bind to the protein. These molecules provide a starting point that facilitates chemical probe development. In these target-based programs, in vitro target-engagement assays are almost always utilized; thus, while these assays may vary in terms of how robust and reliable they are, they are rarely overlooked.

When researchers move into model systems, cultured cells and especially animal models, target-engagement assays become more challenging and are more likely to be omitted. Fortunately, there has been an uptick in the number and types of assays that are suited to this purpose, so scientists have a growing arsenal of assays to choose from to provide convincing evidence that in the system in question, their chemical probe is acting as predicted against the protein of interest. As the Portal grows, we hope to highlight advances in this exciting and important area of assay development to help our users select the best assays available for their protein of interest.

In the meantime, we recommend scientists consider the following papers: Target validation using chemical probes; Determining target engagement in living systems; and Small-molecule target engagement in cells.

Engaging targets