Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.
Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.
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DOI：
10.1021/ja500636p
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发表时间：
2014-04-23
影响因子：
15
通讯作者：
Cunningham BT
中科院分区：
文献类型：
--
作者：
Zhang M;Peh J;Hergenrother PJ;Cunningham BT
高通量筛选使得能够通过完善的比色或荧光活性测定来鉴定重要药物靶点的小分子调节剂。然而，识别非酶蛋白靶标的小分子结合物的现有方法缺乏简单性（例如，需要用报告基因标记结合配偶体之一）或广泛用于 HTS 的酶测定固有的通量。因此，人们对开发用于无标记检测蛋白质-小分子相互作用的高通量技术产生了浓厚的兴趣。在这里，我们描述了一种新型自参考外腔激光（ECL）生物传感器方法，该方法可实现高分辨率和高灵敏度，同时以亚皮米波长精度消除热噪声。使用自参考 ECL 生物传感器，我们演示了小分子与各种固定蛋白靶标之间的结合检测，这些靶标具有从亚纳摩尔到低微摩尔的结合亲和力或抑制常数。最后，对碳酸酐酶同工酶 II 的抑制剂进行“大海捞针”筛选，其中已知的抑制剂与化合物库中的非活性分子明显区分开来。
High-throughput screening has enabled the identification of small molecule modulators of important drug targets via well-established colorimetric or fluorimetric activity assays. However, existing methods to identify small molecule binders of nonenzymatic protein targets lack either the simplicity (e.g., require labeling one of the binding partners with a reporter) or throughput inherent in enzymatic assays widely used for HTS. Thus, there is intense interest in the development of high-throughput technologies for label-free detection of protein–small molecule interactions. Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar. Finally, a “needle-in-the-haystack” screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.
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影响因子：
3
作者：
Makley LN;Gestwicki JE
通讯作者：
Gestwicki JE
影响因子：
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作者：
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通讯作者：
Myszka, DG
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作者：
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通讯作者：
Kodadek, Thomas
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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