A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.
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.
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, United States of America.
Steven B
SB
Ouellette
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, United States of America.
Brett M
BM
Noel
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, United States of America.
Laurie L
LL
Parker
D045744:Cell Line, Tumor; D055808:Drug Discovery; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D016313:Genes, abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity
D010455:Peptides; D047428:Protein Kinase Inhibitors