Quantitative, High-Throughput Cell-Based Assays for Inhibitors of trkA Receptor

Angeles, Thelma S.; Lippy, Jonathan; Yang, Shi

doi:10.1006/abio.1999.4441

Cited by 16 publications

(10 citation statements)

References 21 publications

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“…Using the DELFIA assay, we were able to format an insulin receptor phosphorylation assay into a 96-well plate. The assay was modified from the trkA assay of Angeles et al 7 by eliminating the need to remove medium prior to lysis and eliminating the centrifugation step after lysing the cells. We did find that the addition of the 3× lysis solution resulted in a greater signal than that found with the 1× solution.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a cell-based assay was reported to measure receptor phosphorylation of trkA directly from cell lysates in a 96-well format. 7 In this method, cells in 96-well plates were stimulated, lysed, sonicated, and centrifuged, all within the 96-well plate. The clarified lysate was then transferred to a 96-well plate coated with an antibody directed against the receptor.…”

Section: Introductionmentioning

confidence: 99%

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Validation of a Cell-Based Screen for Insulin Receptor Modulators by Quantification of Insulin Receptor Phosphorylation

et al. 2003

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Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that include phosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor activation. Receptor phosphorylation has been measured using Western blot analysis, which is very low throughput and not easily quantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high throughput. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volume screening in a microwell format. (Journal of Biomolecular Screening 2003:439-446)

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Validation of a Cell-Based Screen for Insulin Receptor Modulators by Quantification of Insulin Receptor Phosphorylation

et al. 2003

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“…The phospho-TrkA ELISA was run from a modified version of a published protocol (34). The MCF10A-TrkA-Δ cells were seeded at a density of 74,000 cells per well and compound treatment was carried out in 10% serum conditions.…”

Section: Kinetic Analysis Of Az-23mentioning

confidence: 99%

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway

Thress

Macintyre

Wang

et al. 2009

Molecular Cancer Therapeutics

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Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer.

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“…Quantitation techniques have been recently developed to increase the throughput of phosphoprotein analysis; however, these techniques require specialized reagents and equipment that can be expensive and limited by availability of compatible reagents [14,15]. Here we describe a rapid, quantitative ELISA-based assay to assess the phosphorylation state of CSF-1R expressed in mammalian cells similar to the previously reported kinase receptor activation ELISA (KIRA-ELISA) method [16,17]. This method has been used to examine the activation and inhibition of CSF-1R overexpressed in human embryonic kidney cells (HEK293E).…”

Section: Introductionmentioning

confidence: 97%

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Baumann¹,

Zeng²,

Donatelli³

et al. 2004

Journal of Biochemical and Biophysical Methods

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Quantitative, High-Throughput Cell-Based Assays for Inhibitors of trkA Receptor

Cited by 16 publications

References 21 publications

Validation of a Cell-Based Screen for Insulin Receptor Modulators by Quantification of Insulin Receptor Phosphorylation

Validation of a Cell-Based Screen for Insulin Receptor Modulators by Quantification of Insulin Receptor Phosphorylation

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

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