Chemical probes are selective modulators that are used in cell assays to link a phenotype to a gene and have become indispensable tools to explore gene function and discover therapeutic targets. While binding to off-targets can be acceptable or beneficial for drugs, it is a confounding factor for chemical probes, as the observed phenotype may be driven by inhibition of an unknown off-target instead of the targeted protein. A negative control - a close chemical analog of the chemical probe that is inactive against the intended target - is typically used to verify that the phenotype is indeed driven by targeted protein. Here, we compare the selectivity profiles of four unrelated chemical probes and their respective negative controls and find that the control is sometimes inactive against up to 80% of known off-targets, suggesting that a lost phenotype upon treatment with the negative control may be driven by loss of inhibition of the off-target. To extend this analysis, we inspect the crystal structures of 90 pairs of unrelated proteins, where both proteins within each pair is in complex with the same drug-like ligand, and estimate that in 50% of cases, methylation (a simple chemical modification often used to generate negative controls) of the ligand at a position that will preclude binding to one protein (intended target) will also preclude binding to the other (off-target). These results uncover a risk associated with the use of negative controls to confirm gene-phenotype associations. We propose that a best practice should rather be to verify that two chemically unrelated chemical probes targeting the same protein lead to the same phenotype.

中文翻译：

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化学探针的阴性对照可能会产生误导

化学探针是一种选择性调节剂，用于细胞测定中以将表型与基因联系起来，已经成为探索基因功能和发现治疗靶标的必不可少的工具。尽管与脱靶结合对于药物而言可能是可接受的或有益的，但这是化学探针的一个混杂因素，因为观察到的表型可能是由未知脱靶而不是靶蛋白的抑制作用所驱动。阴性对照-对预期目标无活性的化学探针的紧密化学类似物-通常用于验证表型确实是由目标蛋白质驱动的。在这里，我们比较了四种不相关的化学探针及其各自的阴性对照的选择性概况，发现该对照有时对多达80％的已知脱靶物无活性，提示用阴性对照治疗后丧失表型可能是由于脱靶抑制作用的丧失所致。为了扩展此分析，我们检查了90对无关蛋白质的晶体结构，其中每对蛋白质均与相同的药物样配体复合，并估计在50％的情况下，甲基化（通常是简单的化学修饰） （用于产生阴性对照）的配体的位置将排除与一种蛋白质（预期的靶标）的结合，也将排除与另一种蛋白质（脱靶的）的结合。这些结果揭示了与使用阴性对照来确认基因表型关联相关的风险。我们建议，最佳实践应该是验证两个针对同一蛋白质的化学无关的化学探针导致相同的表型。