促炎细胞因子
小分子
化学
炎症
蛋白质-蛋白质相互作用
抗体
高通量筛选
重组DNA
生物化学
生物
免疫学
基因
作者
Tiziana Benicchi,Sara Iozzi,Andreas Svahn,Hanna Axelsson,Elisa Mori,Simonetta Bernocco,Federico Cappelli,Chiara Caramelli,Paola Fanti,Eva Genesio,Laura Maccari,Natalia Markova,Iolanda Micco,Valentina Porcari,Johan Schultz,Wolfgang Fecke
标识
DOI:10.1177/1087057112447873
摘要
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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