Abstract
Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments.
Highlights
Protein tyrosine kinases (PTKs) act as central hubs in cellular signaling that tightly control critical cellular functions such as proliferation, apoptosis, and differentiation
In the study by Mand et al, 96-well filter plates were used as a method to prepare K562 cell lysates that were subsequently assayed for Abl kinase activity by incubating the extract with an Abl substrate peptide immobilized on a 96-well, NeutravidinTM coated plate
We found that the lowest Coefficient of variation (CV) occurred at the lowest substrate concentration tested (S1 Fig), and that this correlated with higher signal to noise ratios (S2 Fig)
Summary
Protein tyrosine kinases (PTKs) act as central hubs in cellular signaling that tightly control critical cellular functions such as proliferation, apoptosis, and differentiation. Genetic mutations can alter kinase activity regulation and result in aberrant signaling that promotes disease pathology, most notably cancer [1]. Over 20 tyrosine kinase inhibitors (TKIs) are FDA approved and have varying degrees of clinical success [2]. Despite TKIs often being the best option for patients, heterogeneous response and acquired resistance remain a significant clinical and economic burden. Drug developers are working to overcome these problems by commercializing generation inhibitors with increased potency, different modes of inhibition, and strategic inhibition of multiple kinases.
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