A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction
A Homogeneous HTRF Assay for the Identification of Inhibitors of the TWEAK-Fn14 Protein Interaction
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DOI：
10.1177/1087057112447873
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发表时间：
2012-08-01
影响因子：
--
通讯作者：
Fecke, Wolfgang
中科院分区：
文献类型：
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作者：
Benicchi, Tiziana;Iozzi, Sara;Fecke, Wolfgang
TWEAK-Fn 14通路在炎症、自身免疫性疾病和癌症模型中上调。TWEAK和Fn 14均显示在CNS中响应于不同刺激（特别是星形胶质细胞、小胶质细胞和神经元）的表达增加，导致NF-κ B的活化和促炎细胞因子的释放。虽然针对这些蛋白质的中和抗体已被证明在炎症的动物模型中具有治疗功效，但还没有可用的小分子治疗剂。在这里，我们描述了一种新的均相时间分辨荧光（HTRF）为基础的筛选试验的发展，连同几个counterassays用于识别这种蛋白质-蛋白质相互作用的小分子抑制剂。使用重组HIS-TWEAK和Fn 14-Fc蛋白以及FLAG-TWEAK和Fn 14-FLAG蛋白和抗Fn 14抗体来建立和验证这些测定，并筛选60000种化合物的文库。两个HTRF counterassay与不相关的蛋白质在相同的测定格式，抗聚集测定和氧化还原测定，被施加到过滤出潜在的假阳性化合物。新的检测和相关的筛选级联应该是有用的TWEAK-Fn 14蛋白相互作用的小分子抑制剂的发现。
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-kappa B 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.