A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions.
A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions.
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DOI：
10.1016/j.ab.2012.09.010
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发表时间：
2012-12-15
影响因子：
2.9
通讯作者：
Jardetzky TS
中科院分区：
文献类型：
--
作者：
Kim B;Tarchevskaya SS;Eggel A;Vogel M;Jardetzky TS
免疫球蛋白E （IgE）抗体与高亲和受体FcεRI的相互作用在引发大多数过敏反应中起着核心作用。ige受体相互作用已成为治疗过敏性疾病的靶标，许多高亲和力的大分子抑制剂已被发现。小分子抑制剂将比目前的抗ige治疗提供显著的优势，但没有候选化合物被确定并充分验证。在这里，我们报告了一个时间分辨荧光共振能量转移（TR-FRET）测定监测ige受体相互作用的发展。当供体镧系荧光团被招募到位点特异性Alexa Fluor 488标记的IgE-Fc和his标记的FcεRIα蛋白复合物中时，TR-FRET测定了荧光强度的增加。该方法可以在平衡竞争结合实验中监测经典的与IgE-Fc或FcεRIα结合的竞争性抑制剂。此外，TR-FRET分析还可用于跟踪IgE-Fc-FcεRIα解离动力学，并识别加速预形成复合物解离的抑制配体，如工程DARPin（设计的锚蛋白重复序列蛋白）抑制剂。TR-FRET测定适用于高通量筛选（HTS），如384孔板格式的美国国立卫生研究院（NIH）临床馆藏库的试点筛选所示。
The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE–receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR–FRET) assay for monitoring the IgE–receptor interaction. The TR–FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR–FRET assay can also be used to follow the kinetics of IgE-Fc–FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR–FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.
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影响因子：
5.6
作者：
Eggel, Alexander;Baumann, Michael J.;Vogel, Monique
通讯作者：
Vogel, Monique
影响因子：
64.5
作者：
Garman, SC;Kinet, JP;Jardetzky, TS
通讯作者：
Jardetzky, TS
影响因子：
2.9
作者：
Henry, AJ;McDonnell, JM;Gould, HJ
通讯作者：
Gould, HJ
影响因子：
4.8
作者：
Hunt, J;Beavil, RL;Beavil, AJ
通讯作者：
Beavil, AJ
影响因子：
16.8
作者：
Holdom MD;Davies AM;Nettleship JE;Bagby SC;Dhaliwal B;Girardi E;Hunt J;Gould HJ;Beavil AJ;McDonnell JM;Owens RJ;Sutton BJ
通讯作者：
Sutton BJ