Cited by 13NF-κB Signaling PathwaysCytokine Signaling Pathways and InteractionsMonoclonal and Polyclonal Antibodies Research

A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction

Open access
Benicchi, Tiziana, Iozzi, Sara, Svahn, Andreas, Axelsson, Hanna, Mori, Elisa, Bernocco, Simonetta, Cappelli, Federico, Caramelli, Chiara +8 more·2012·SLAS Discovery

Abstract

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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Crossref · 10.1177/1087057112447873

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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
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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