Achieving the promise and avoiding the peril of chemical probes using genetics

Chemical probes can be valuable tools for studying protein targets, but addressing concerns about a probe's cellular target or its specificity can be challenging. A reliable strategy is to use a mutation that does not alter a target's function but confers resistance (or sensitizes) to the inhibitor in both cellular and biochemical assays. However, challenges remain in finding such mutations. Here, we discuss structure- and cell-based approaches to identify resistance- and sensitivity-conferring mutations. Further, we describe how resistance-conferring mutations can help with compound design, and the use of saturation mutagenesis to characterize a compound binding site. We highlight how genetic approaches can ensure the proper use of chemical inhibitors to pursue mechanistic studies and test therapeutic hypotheses.