Cell treatment and lysis in 96-well filter-bottom plates for screening Bcr-Abl activity and inhibition in whole-cell extracts.
Although conventional high-throughput screens performed in vitro with purified protein kinases are powerful tools to discover new kinase inhibitors, they are far from ideal for determining efficacy in vivo. As a complementary approach, cell-based, target-driven secondary screens may help predict in vivo compound potency and specificity as well as evaluate bioavailability and toxicity. Here the authors report a simple protocol for treating K562 Bcr-Abl-expressing cells with small-molecule kinase inhibitors in 96-well filter-bottom plates followed by in-plate cell lysis. The lysates were assayed via a solid-phase kinase assay, allowing determination of apparent IC(50) for known Bcr-Abl inhibitors as well as facilitating the screening of a small kinase inhibitor library. This approach may have further applications in generating lysates for analyzing kinase activity and inhibition in other nonadherent suspension cell lines.
Mand
Michael R
MR
Institute of Cellular and Molecular Biology, University of Texas at Austin, USA.
Wu
Ding
D
Veach
Darren R
DR
Kron
Stephen J
SJ
D001549:Benzamides; D002457:Cell Extracts; D002458:Cell Fractionation / Q000295:instrumentation* / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors*; D006801:Humans; D000068877:Imatinib Mesylate; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D008954:Models, Biological; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
D001549:Benzamides; D002457:Cell Extracts; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D054852:Small Molecule Libraries; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl