Here, we establish that chaperones can be used as ‘thermodynamic sensors’ for drug–target interactions. We use HSP90, CDC37 and HSC70 as examples of chaperones, and kinases and steroid hormone receptors as examples of drug targets. But it is likely that our approach will prove broadly applicable with other chaperones and other drug targets. The binding of small molecules to their targets generally leads to thermodynamic stabilization of those targets40
. If the target proteins are in equilibrium between their fully folded and partially folded chaperone-bound conformations, drug binding will lead to decreased chaperone association. Chaperones associate with a large fraction of proteins in all organisms even at their steady states1, 41, 42
, and, as we have shown, proteins that do not associate with them in this manner can be readily engineered to do so.
Our assay does not depend on the enzymatic activity of the target protein nor on a particular binding pocket in the target. Thus, it should be well suited for the pursuit of drugs for targets that have been notoriously difficult to assay in biologically relevant contexts, including E3 ligases1
, transcription factors1
, and diverse protein–protein interactions. Our method is amenable to high-throughput automation, bridging the gap between the more traditional high-throughput in vitro
methods and lower throughput in vivo
approaches in drug discovery.
We illustrated the utility of chaperone profiling by surveying kinase inhibitor specificities in vivo
. Compared to other available methods for kinase inhibitor profiling5–7
, our approach has several advantages. First, it uses full-length kinases in their native cellular environment, in the context of physiological post-translational modifications, conformational switches, and cellular interactors. Although the expression levels of the kinases in our assay are higher than endogenous levels, the strong correlation between our results and in vivo
potency suggests that this is not a generally confounding factor. With the use of lentiviral delivery systems it should be readily adaptable to any cell type in cases where a particular cellular context is required (e.g. stem cells, neuronal cells). Second, kinases are assayed at physiological ATP concentrations. As most kinase inhibitors are ATP-competitive, the difference between in vitro
and in vivo
ATP concentrations often leads to substantial changes in cellular potency. Third, the assay is compatible with allosteric inhibitors and activators, in contrast to other in vivo
. For example, neither GNF-2 nor MK2206 would have been identified in an in vitro
screen using ABL1 or AKT1 kinase domains only, as the inhibitors absolutely require the kinases’ regulatory domains for function27
Finally, we established that the assay can be used to discover clinically relevant drug targets. NTRK3 and its translocation fusion variant ETV6-NTRK3 proved to be prominent targets of crizotinib. Crizotinib inhibits ETV6-NTRK3 with nanomolar potency and treatment of ETV6-NTRK3-dependent xenografts with crizotinib leads to dramatic regression of tumors. These results suggest that it might be worth testing crizotinib as a therapeutic agent against cancers driven by the ETV6-NTRK3 translocation. Crizotinib already is already approved for use in the treatment of certain lung cancers. Recommended dosing parameters with a good safety profile for children have already been established43
. Thus it should be possible to translate these findings rapidly to efficacy trials in selected pediatric malignancies such as infantile fibrosarcoma. Furthermore, given the intense interest in repurposing drugs and expanding the scope of druggable targets, the ease and reproducibility of our assay should have broad application in drug development.