A homogeneous HTRF assay for the identification of inhibitors of the TWEAK-Fn14 protein interaction.
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.
Biomolecular Screening Unit, Siena Biotech S.p.A, Siena, Italy.
Tiziana
T
Benicchi
Sara
S
Iozzi
Andreas
A
Svahn
Hanna
H
Axelsson
Elisa
E
Mori
Simonetta
S
Bernocco
Federico
F
Cappelli
Chiara
C
Caramelli
Paola
P
Fanti
Eva
E
Genesio
Laura
L
Maccari
Natalia
N
Markova
Iolanda
I
Micco
Valentina
V
Porcari
Johan
J
Schultz
Wolfgang
W
Fecke
D001327:Autoimmune Diseases / Q000378:metabolism; D002460:Cell Line; D000074049:Cytokine TWEAK; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000074050:TWEAK Receptor; D000079424:Tumor Necrosis Factor Inhibitors; D048069:Tumor Necrosis Factors / Q000378:metabolism
D000074049:Cytokine TWEAK; D009842:Oligopeptides; D010455:Peptides; D018124:Receptors, Tumor Necrosis Factor; C000613297:TNFRSF12A protein, human; C109743:TNFSF12 protein, human; D000074050:TWEAK Receptor; D000079424:Tumor Necrosis Factor Inhibitors; D048069:Tumor Necrosis Factors; C085706:FLAG peptide