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Updated: Jun 8, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
High-throughput molecular imaging for the identification of FADD kinase inhibitors
Amjad P Khan1, Katrina A Schinske, Shyam Nyati
1Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
Fas-associated protein with death domain (FADD) was originally reported as a proapoptotic adaptor molecule that mediates receptor-induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Overexpression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, the authors developed a cell-based reporter for FADD kinase activity. The reporter assay, optimized for a high-throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
Insights
Researchers developed a novel cell-based reporter assay to screen for compounds targeting Fas-associated protein with death domain (FADD) kinase activity, a potential cancer therapeutic target.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Biology
Background:
- Fas-associated protein with death domain (FADD) is an adaptor molecule primarily known for its role in apoptosis.
- Emerging evidence implicates FADD in critical cellular processes including NF-κB activation, cell cycle regulation, and proliferation.
- Aberrant FADD expression and phosphorylation correlate with cancer phenotypes, highlighting FADD as a promising therapeutic target.
Purpose of the Study:
- To elucidate signaling pathways regulating FADD phosphorylation.
- To identify novel small molecules that inhibit FADD kinase activity.
- To develop a high-throughput screening (HTS) assay for FADD kinase activity.
Main Methods:
- Development of a cell-based bioluminescence reporter assay for FADD kinase activity.
- Optimization of the reporter assay for high-throughput screening (HTS).
- Demonstration of the reporter cell line's utility in evaluating pharmacologic properties in mouse models.
Main Results:
- A functional cell-based reporter assay for FADD kinase activity was successfully established and optimized for HTS.
- The reporter system enables live-cell measurement of FADD kinase modulation.
- The reporter cell line facilitates rapid preclinical evaluation of drug candidates in vivo.
Conclusions:
- The developed reporter assay provides a valuable tool for discovering FADD-targeting therapeutics.
- This platform can accelerate the identification and validation of novel anti-cancer compounds.
- The FADD signaling pathway represents a viable target for cancer therapy development.
