Real-time monitoring of kinase activity using luminescent chemical probes

Kinase enzymes represent one of the most promising targets for drug discovery in oncology. The search for new kinase inhibitors requires robust, sensitive, affordable assays for kinase activity. Most commercial kinase assays are restricted to end-point measurements, which give no kinetic or mechanistic information and require either expensive antibodies to recognise the phosphopeptide or chemically modified substrates. Crucially, there is no low-cost method available for continuous measurement of kinase activity, which limits our understanding of kinase kinetics.

We will create probes that provide a unique luminescent signal that precisely indicates the ratio of ATP/ADP in real time. We will develop luminescent probes that can distinguish between ADP and ATP at physiological pH, thereby allowing continuous measurement of the kinase-catalysed conversion of ATP to ADP. We will harness the long luminescence lifetime of europium probes to enable time-gated detection and removal of autofluorescence from peptides and proteins. We will also measure the activity of selected kinases by varying the amount of enzyme, substrate and co-factor systematically. This will require optimisation of probe sensitivity and calibration of luminescent response.

Once these parameters have been optimised, we will use our assay to examine selective inhibitors of kinase activity. Our findings could help with drug discovery in oncology.