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.
Institute of Cellular and Molecular Biology, University of Texas at Austin, USA.
Michael R
MR
Mand
Ding
D
Wu
Darren R
DR
Veach
Stephen J
SJ
Kron
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