Kinase inhibitors created a new paradigm in chemotherapy and are a major focus of new oncology drugdevelopment, but developmental success rates are low (5-10%). Resistance to kinase inhibitors occurs throughtarget-dependent mechanisms (e.g. point mutations that abrogate drug binding) and target-independentmechanisms (e.g. upregulation of alternative signaling pathways, termed 'kinome reprogramming').Therefore, combinations of inhibitors that target several kinases at once are desirable to have a better chance ofavoiding the resistance/relapse cycle. Detecting protein kinase activity inside living cells (rather than in lysatesor reconstituted systems) is important for understanding kinase inhibitor drug sensitivity and resistancemechanisms, and would lead to better screening for inhibitors likely to make it through the developmentprocess. We will develop multiplexed, cell-based assays for specific kinase activities that are important toinhibitor response and kinome reprogramming, and use them to detect kinase activation profiles in patientcells and for inhibitor screens. Phosphorylation of the biosensors is detected using time-resolved lanthanideluminescence measurements, in which Tb3+ emission energy is measured directly via small moleculefluorophores to give different emission colors depending on the fluorophore.
In Aim 1, we will establishquantitative assays for activity (and therefore inhibition) of key kinases in drug sensitive and drug resistantCML cells, profiling the pathway activation phenotypes in therapeutically relevant cellular states. We will usethe set of biosensors we have already established in preliminary work, and add biosensors for other kinases asthey are developed. The assays will be established with cell lines and samples from CML patients (comparing tohealthy controls), and validated with RT-qPCR and SWATH' proteomics.
In Aim 2, we will screen forsynergies between existing kinase inhibitor drugs and new compounds (via libraries). We will also develophomogenous multiplexed analysis of biosensor phosphorylation using energy transfer from lanthanides toorganic dyes.
In Aim 3, we expand the biosensor design pipeline to develop new, non-natural peptidesubstrates to use as biosensors for other kinases. The work described in this aim will add to the set ofbiosensors we already have available. Completion of the work described in this proposal will give us a novelassay for multiple kinases, a suite of new biosensors as well as new and refined detection strategies to use inscreening. Drug discovery will benefit from this technology's ability to address kinome reprogrammingmechanisms by targeting several kinases at a time. Drug development will benefit from companion assays formultiple targets that could follow a drug or drug combination through the hit to lead transition, targetvalidation, pre-clinical studies, clinical trials, and beyond into treatment management. This assay and itsassociated tools will contribute to the next generation of targeted therapy development in cancer by breakingnew ground in our ability to model the disease environment during drug screening and development.
Kinase inhibitor drugs have changed the face of chemotherapy and are projected to remain a major focus of leukemia treatment; however; their long-term efficacy is variable because of biological complexity and resistance. We will develop an assay strategy that can be used to screen new drugs and drug combinations in a way that better mimics biological complexity. This assay could also be used as a companion diagnostic to monitor the effectiveness of treatment; finding resistance earlier so patients can try alternatives that could improve their treatment outcomes.
