Authors

Discussion

Small-molecule kinase inhibitors are an important cancer therapy approach, many targeting the highly conserved ATP binding pocket aiming to block ATP interaction with the kinase. However, low selectivity and development of gatekeeper mutations ultimately leads to acquired resistance, which is driving a new search for kinase inhibitors with an allosteric mechanism of action. Type III kinase inhibitors bind to target kinases allosterically within the catalytic domain, adjacent to the ATP binding-site. Once bound, these inhibitors have been observed to allow and promote the binding of ATP. We leveraged this feature, using a fluorescent ATP analogue, to develop an assay to detect type III inhibitors.

Using a known MEK1 type III kinase inhibitor – PD0325901 (mirdametinib) - an inhibitor-dependent increase in ATP binding was measured in an HTRF assay. When the fluorescent ATP and labelled anti-GST antibody simultaneously bind to GST-tagged MEK1, the proximity of the two fluorophores allows a FRET signal to be established. Following buffer optimisation, more than a 10-fold increase in signal was established when applying an ATP titration in the presence of PD0325901 when compared to MEK1 alone. Dose-ratio experiments with respect to both fluorescent ATP and inhibitor, showed that PD0325901 application induced a leftward shift in KD meaning the inhibitor is altering the molecular binding pharmacology. The same was true in respect to the inhibitor titration; increasing ATP concentration induced a leftward shift in PD0325901 EC50. 

Given these promising results, we aim to apply this assay to the detection of further type III allosteric inhibitors, particularly for kinase targets currently without reference type III inhibitor compounds, and to potentially identify inhibitors with an allosteric mode of action in our future projects.