Negative controls of chemical probes can be misleading

Abstract

ABSTRACTChemical 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.Abstract Figure