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High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)-protein tyrosine kinase
Eric A Kumar1, Casey D Charvet, G L Lokesh
1Eppley Institute for Cancer Research and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Abstract:
The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library.
Insights
Casitas B-lineage lymphoma (Cbl) proteins regulate signaling pathways. Researchers developed a high-throughput assay to find inhibitors of Cbl tyrosine kinase binding (TKB) domains, potentially targeting cancers and inflammatory diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Casitas B-lineage lymphoma (Cbl) proteins are E3 ubiquitin ligases regulating signal transduction.
- Cbl proteins, via their tyrosine kinase binding (TKB) domain, control cell surface protein tyrosine kinase (PTK) levels.
- Dysfunctional Cbl signaling contributes to inflammatory diseases and cancers due to prolonged PTK activation.
Purpose of the Study:
- To develop a high-throughput fluorescence polarization assay for identifying inhibitors of the Cbl(TKB) domain.
- To explore Cbl proteins as potential therapeutic targets in cancer and inflammatory conditions.
Main Methods:
- Development of a competitive high-throughput fluorescence polarization assay.
- Assay implemented in a 384-well format for screening.
- Screening of the Prestwick Chemical Library to validate assay readiness.
Main Results:
- Successful development of a high-throughput fluorescence polarization assay for Cbl(TKB) inhibitors.
- Demonstrated the assay's readiness for large-scale screening.
- Identified potential inhibitors from the Prestwick Chemical Library.
Conclusions:
- The developed assay is suitable for identifying inhibitors of Cbl(TKB).
- Cbl proteins represent a promising, yet under-explored, therapeutic target for cancers and inflammatory diseases.
- Further research into Cbl inhibitors could lead to novel treatments.
