Cell treatment and lysis in 96-well filter-bottom plates for screening Bcr-Abl activity and inhibition in whole-cell extracts Journal Article
| Authors: | Mand, M. R.; Wu, D.; Veach, D. R.; Kron, S. J. |
| Article Title: | Cell treatment and lysis in 96-well filter-bottom plates for screening Bcr-Abl activity and inhibition in whole-cell extracts |
| Abstract: | 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, targetdriven 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. © 2010 Society for Biomolecular Sciences. |
| Keywords: | controlled study; protein expression; unclassified drug; drug targeting; sensitivity and specificity; reproducibility of results; imatinib; enzyme inhibition; models, biological; protein kinase inhibitor; culture medium; drug potency; high throughput screening; drug screening; dose-response relationship, drug; small molecule libraries; 6 (2,6 dichlorophenyl) 2 [3 (hydroxymethyl)anilino] 8 methylpyrido[2,3 d]pyrimidin 7(8h) one; dasatinib; pyrimidines; prediction; protein kinase inhibitors; cell fractionation; cytolysis; bcr abl protein; piperazines; ic 50; inhibitory concentration 50; suspension cell culture; enzyme assay; fusion proteins, bcr-abl; cell extracts; bcr-abl; 6 (2,6 dichlorophenyl) 2 (4 fluoro 3 methylanilino) 8 methyl 8h pyrido[2,3 d]pyrimidin 7 one; 6 (2,6 dichlorophenyl) 8 methyl 2 (3 methylthioanilino) 8h pyrido[2,3 d]pyrimidin 7 one; pd 173952; k562 cells; cell lysis; lead identification; secondary assay; dv 1 10; dv 2 271; dv 2 273; dv 2 281; dv 2 289; dv 2 37; dv 2 43; dv 2 47; dv 2 89; dv 3 081; dv 3 096; dv 5 31; cell strain k 562 |
| Journal Title: | Journal of Biomolecular Screening |
| Volume: | 15 |
| Issue: | 4 |
| ISSN: | 1087-0571 |
| Publisher: | Sage Publications |
| Date Published: | 2010-04-01 |
| Start Page: | 434 |
| End Page: | 440 |
| Language: | English |
| DOI: | 10.1177/1087057110363307 |
| PUBMED: | 20237206 |
| PROVIDER: | scopus |
| PMCID: | PMC4471859 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 20 April 2011" - "CODEN: JBISF" - "Source: Scopus" |
