Identifying the direct physiological targets of drugs and chemical probes remains challenging. Here we describe how resistance can be used to achieve 'gold-standard' validation of a chemical inhibitor's direct target in human cells. This involves demonstrating that a silent mutation in the target that suppresses inhibitor activity in cell-based assays can also reduce inhibitor potency in biochemical assays. Further, phenotypes due to target inhibition can be identified as those observed in the inhibitor-sensitive cells, across a range of inhibitor concentrations, but not in genetically matched cells with a silent resistance-conferring mutation in the target. We propose that chemotype-specific resistance, which is generally considered to be a limitation of molecularly targeted agents, can be leveraged to deconvolve the mechanism of action of drugs and to properly use chemical probes.
Keywords: chemical biology; chemical probes; drug resistance; target identification.
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