A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction
Open accessAbstract
The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.
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- Authors
- Benicchi, T., Iozzi, S., Svahn, A., Axelsson, H., Mori, E., Bernocco, S., Cappelli, F., Caramelli, C., Fanti, P., Genesio, E., Maccari, L., Markova, N., Micco, I., Porcari, V., Schultz, J., & Fecke, W.
- Affiliations
- Siena Biotech (Italy) (IT)
- Corresponding author
- Fecke, Wolfgang
- Venue
- SLAS Discovery
- Publisher
- Elsevier BV
- Published
- 2012-08
- Volume
- 17
- Issue
- 7
- Pages
- 933-945
- ISSN
- 2472-5552 (ISSN-L)
- OpenAlex
- W2025463155
- Cited by
- 12
- References
- 39
- Citations by year
- 2022: 1 · 2021: 1 · 2020: 1 · 2018: 1 · 2015: 3 · 2014: 1 · 2013: 4
- FWCI
- 0.85
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- SLAS DISCOVERY (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
- Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Keywords
- TWEAK, Fn14, protein-protein interaction, small molecule inhibitors, homogeneous time-resolved fluorescence, high-throughput screening, false positive reduction, inflammation, NF-κB, inflammatory cytokines, central nervous system
- MeSH terms
- Cytokine TWEAK, TWEAK Receptor, Tumor Necrosis Factor Inhibitors, Autoimmune Diseases, Cell Line, Drug Screening Assays, Antitumor, Humans, Inflammation, Neoplasms, Oligopeptides, Peptides, Receptors, Tumor Necrosis Factor, Tumor Necrosis Factors, High-Throughput Screening Assays, HEK293 Cells
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