Zhang Meng, Peh Jessie, Hergenrother Paul J, Cunningham Brian T
Department of Physics, ‡Department of Chemistry, §Department of Bioengineering, and ⊥Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
J Am Chem Soc. 2014 Apr 23;136(16):5840-3. doi: 10.1021/ja500636p. Epub 2014 Apr 10.
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.
高通量筛选能够通过成熟的比色或荧光活性测定法鉴定重要药物靶点的小分子调节剂。然而，现有的鉴定非酶蛋白靶点小分子结合剂的方法，要么缺乏简便性（例如，需要用报告分子标记其中一个结合伴侣），要么缺乏广泛用于高通量筛选的酶促测定法所固有的通量。因此，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引起了人们的浓厚兴趣。在此，我们描述了一种新型的自参考外腔激光（ECL）生物传感器方法，该方法实现了高分辨率和高灵敏度，同时以亚皮米波长精度消除了热噪声。使用自参考ECL生物传感器，我们证明了能够检测小分子与多种固定化蛋白靶点之间的结合，这些靶点对的结合亲和力或抑制常数范围从亚纳摩尔到低微摩尔。最后，针对碳酸酐酶同工酶II进行了“大海捞针”式的抑制剂筛选，其中已知抑制剂能够与化合物库中的无活性分子明显区分开来。
