A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases
A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases
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DOI：
10.1021/ja104491h
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发表时间：
2010-08-25
影响因子：
15
通讯作者：
Ghosh, Indraneel
中科院分区：
文献类型：
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作者：
Jester, Benjamin W.;Cox, Kurt J.;Ghosh, Indraneel
人类基因组中编码的 518 种蛋白激酶受到精细调控，它们的异常功能通常与人类疾病相关。因此，为了推进治疗并探测信号转导级联，人们对开发可以选择性靶向蛋白激酶的抑制剂非常感兴趣。然而，利用传统的活性测定法很难常规地识别针对如此大量的蛋白激酶的特定化合物，其中需要纯化的蛋白激酶。为了寻找一种简单、快速、实用的方法来识别特定的抑制剂，我们描述了利用卷曲螺旋辅助三杂交系统的分裂蛋白方法的开发和应用。在这种方法中，目标蛋白激酶附着在分裂萤火虫荧光素酶的 C 末端片段上，而卷曲螺旋 Fos（卷曲螺旋 Jun 特有的）附着在 N 末端片段上。添加与全激酶抑制剂（例如十字孢菌素）缀合的 Jun 后，会建立三杂交复合物，并伴随分裂荧光素酶的重新组装。可以通过修饰星形孢菌素的置换导致分裂荧光素酶活性的相应损失来潜在地鉴定抑制剂。我们通过测试不同激酶家族的蛋白激酶证明了这种新的三杂交方法具有潜在的通用性。为了询问该方法是否允许筛选抑制剂，我们针对 80 种已知蛋白激酶抑制剂的库测试了六种不同的蛋白激酶。最后，我们证明这种三混合系统有可能为结构/功能分析提供一种快速方法，并有助于识别变构抑制剂。
The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades, there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil-assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors.