The Promise and Peril of Chemical Probe Negative Controls

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. Chemical
probe off-targets are a confounding factor 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 the targeted
protein. Here, we compare the selectivity profiles of four unrelated
chemical probes and their respective negative controls. We find that
controls that chemically deviate from the probe by a single heavy
atom can be inactive against up to 80% of known off-targets if the
chemical modification has a charge-neutralizing effect. In such cases,
a loss in phenotype upon treatment with the negative control may be
driven by loss of inhibition of an off-target. To expand 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. We computationally estimate that in 50% of cases, methylation
of the ligand (a simple chemical modification often used to generate
negative controls) at a position that will preclude binding to one
protein (the intended target) will also preclude binding to the other
(the off-target). These results emphasize the need to select negative
controls with care and profile both chemical probes and negative controls
against diverse protein arrays to verify that off-targets of probes
are also hit by negative controls. When available, a best practice
should be to verify that two unrelated chemical probes targeting the
same protein elicit the same phenotype.