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.

确定药物和化学探针的直接生理目标仍然具有挑战性。在这里，我们描述了抗性如何用于实现人体细胞中化学抑制剂直接靶标的“金标准”验证。这涉及证明靶标中的沉默突变在基于细胞的测定中抑制抑制剂的活性也可以降低生化测定中的抑制剂效价。此外，可以将归因于靶标抑制的表型鉴定为在一系列抑制剂浓度范围内的抑制剂敏感细胞中观察到的表型，但不能在靶中具有沉默抗性突变的基因匹配细胞中观察到。我们提出化学型特异性抗药性，通常被认为是分子靶向药物的局限性，