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Updated: Aug 19, 2026
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the IN Cell 3000 analyzer
Betina Kerstin Lundholt1, Viggo Linde, Frosty Loechel
1BioImage A/S, Copenhagen, Denmark.
Abstract:
The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization.
Insights
Researchers developed a novel assay to find inhibitors of the PI3-kinase/Akt pathway, crucial for cancer survival. This screening identified new Akt1 translocation inhibitors, offering potential therapeutic strategies for cancer treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- The Phosphatidylinositol 3-kinase (PI3K)/Akt pathway is vital for cell survival and frequently dysregulated in human cancers.
- Targeting this pathway is a key strategy in cancer therapy, but specific catalytic inhibitors for Akt1 remain elusive.
- Akt1 activation involves translocation, presenting a potential target for drug development.
Purpose of the Study:
- To develop and validate a novel fluorometric imaging plate reader (FLIPR)-based assay for detecting Akt1 activation.
- To screen a large chemical library to identify inhibitors of Akt1 translocation.
- To characterize the mechanism of action of identified Akt1 translocation inhibitors.
Main Methods:
- Development of a FLIPR-based assay to monitor Akt1 translocation.
- High-throughput screening of a 45,000-compound chemical library.
- Utilized in vitro assays and pathway-specific translocation assays for inhibitor characterization.
Main Results:
- Successful identification of multiple classes of Akt1 translocation inhibitors from the chemical library.
- Characterization of the mechanisms of action for two distinct chemical classes of inhibitors.
- Demonstrated the utility of protein translocation assays in hit identification and characterization.
Conclusions:
- Protein translocation assays are effective tools for discovering and characterizing inhibitors of signaling pathways like Akt1.
- The identified Akt1 translocation inhibitors represent promising leads for developing novel cancer therapeutics.
- This work provides a foundation for further drug development targeting the PI3K/Akt pathway in cancer.
