Cell Treatment and Lysis in 96-Well Filter-Bottom Plates for Screening Bcr-Abl Activity and Inhibition in Whole-Cell Extracts

Mand, Michael R.; Wu, Duojie; Veach, Darren R.; Kron, Stephen J.

doi:10.1177/1087057110363307

Cited by 4 publications

(6 citation statements)

References 25 publications

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“…The kinase activity IC 50 values for imatinib, nilotinib, and dasatinib were 150 nM (95% CI, 100–223 nM), 10 nM (95% CI, 4–25 nM), and 1 nM (95% CI, 0.5–1.2 nM), respectively ( Fig 3B ). These values were within 10-fold of the biochemical and cytotoxicity IC 50 values reported in the literature, and comparable to IC 50 values for the same TKIs using K562 cell lysates [ 33 , 35 ] However, activity- and cytotoxic-based inhibition assays should be used as complementary approaches for measuring drug action. Cytotoxicity (i.e.…”

Section: Resultssupporting

confidence: 81%

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A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

2016

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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.

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Section: Resultssupporting

confidence: 81%

“…Streamlining the assay for multi-well plates was inspired by a workflow described by Mand and colleagues [ 33 ]. The method uses a 96-well filter plate for cell treatment and lysis, followed by ELISA-based detection of substrate phosphorylation ( Fig 1 ).…”

Section: Resultsmentioning

confidence: 99%

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

2016

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“…Compared to their activity in lysates, PD166326 and dasatinib demonstrated a significant increase in potency in cells (Figure 5B). These results were surprising although this increase in potency was observed in previous studies (18). Several factors may explain our results.…”

Section: Resultscontrasting

confidence: 50%

“…On the other hand, inhibition in cultured K-562 cells was performed in 96-well filter plates, where cells were dosed, lysed, and harvested in-plate. Although details of the approach are described and validated elsewhere (18), the inhibition of kinases in cultured cells involved the distribution of confluent cells in sterile 96-well filter plates and incubation with inhibitors for one hour. Cells were washed by centrifugation, lysed in-plate, and lysates collected by a second 96-well plate during centrifugation at 4 °C.…”

Section: Resultsmentioning

confidence: 99%

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A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells

Sylvester

Kron

2010

Molecular Cancer Therapeutics

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Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from wellspecific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.

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Photocleavable Peptide-Conjugated Magnetic Beads for Protein Kinase Assays by MALDI-TOF MS

Zhou

Yan

et al. 2010

Bioconjugate Chem.

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Peptides were immobilized onto superparamagnetic beads via photocleavable linkers. This enabled simple, rapid, and label-free protein kinase assays via MALDI-TOF MS detection of substrate peptide phosphorylation. Abltide, a model substrate for the Abl protein tyrosine kinase model, was coupled onto amine-terminated beads, incubated with ATP and recombinant c-Abl kinase, and released and further detected to determine phosphorylation. Abltide phosphorylation was found to depend significantly on the length and composition of linkers to the bead surface. Inserting a diblock spacer of poly(glycine) and poly(ethylene glycol) segments markedly enhanced phosphorylation. To validate the assay, the activity of two small-molecule kinase inhibitors, imatinib and dasatinib, which target the oncogenic mutant tyrosine kinase Bcr-Abl to treat chronic myeloid leukemia (CML), was tested. Examining inhibition of the purified c-Abl or Bcr-Abl in K562 CML cell extracts, IC50 values were determined to be consistent with the literature. This simple, label-free, MALDI-based protein kinase assay can be readily adapted to allow multiplexed assays of multiple peptide substrates and/or analysis of alternative post-translational modifications as a tool for drug discovery and clinical testing.

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Cell Treatment and Lysis in 96-Well Filter-Bottom Plates for Screening Bcr-Abl Activity and Inhibition in Whole-Cell Extracts

Cited by 4 publications

References 25 publications

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells

Photocleavable Peptide-Conjugated Magnetic Beads for Protein Kinase Assays by MALDI-TOF MS

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