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Microtiter cell-based assay for detection of agents that alter cellular levels of Her2 and EGFR
Henri Huezo1, Maria Vilenchik, Neal Rosen
1Program in Cell Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Abstract:
Overexpression of the transmembrane tyrosine kinases Her2 and EGFR is associated with aggressive malignancies, and several therapeutic strategies targeting the two receptors are now in various stages of clinical development. Most of the known agents block the activation or inhibit the activity of the kinases; however, a more significant therapeutic outcome may result from degrading these oncoproteins. Here, we report the development of a microtiter cell-based assay that sensitively detects cellular levels of Her2 and EGFR. The assay is useful in identifying small molecules that alter cellular levels of these kinases and in quantifying their effect. The method gives results comparable to Western blot, but it is faster, less labor intensive, and amenable to high throughput.
Insights
We developed a new assay to measure levels of Her2 and EGFR proteins in cells. This high-throughput method helps find drugs that can degrade these oncoproteins, potentially improving cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Assay Development
Background:
- Overexpression of Her2 and EGFR tyrosine kinases is linked to aggressive cancers.
- Current therapies primarily inhibit kinase activity, but degrading these oncoproteins may offer better outcomes.
Purpose of the Study:
- To develop a novel cell-based assay for quantifying cellular Her2 and EGFR levels.
- To identify small molecules capable of reducing these oncoprotein levels.
Main Methods:
- A microtiter plate assay was developed to detect cellular Her2 and EGFR.
- The assay was used to screen for small molecules affecting oncoprotein levels.
- Results were compared to traditional Western blot analysis.
Main Results:
- The assay sensitively detects cellular Her2 and EGFR levels.
- It successfully identified small molecules that alter these kinase levels.
- The assay provides results comparable to Western blot but is faster and high-throughput.
Conclusions:
- A new, efficient cell-based assay for Her2 and EGFR has been developed.
- This assay facilitates the discovery of drugs targeting oncoprotein degradation.
- The method offers a high-throughput alternative to Western blotting for kinase quantification.
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