A detergent-based assay for the detection of promiscuous inhibitors
A detergent-based assay for the detection of promiscuous inhibitors
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DOI：
10.1038/nprot.2006.77
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Shoichet, Brian K.
中科院分区：
文献类型：
--
作者：
Feng, Brian Y.;Shoichet, Brian K.
在微摩尔浓度下，许多小分子自缔合成非特异性抑制酶和其他蛋白质的胶体聚集体。在这里，我们描述了一个协议，用于识别聚集为基础的抑制剂，并区分它们从小分子抑制通过特定的机制。作为混杂的、基于聚集体的抑制的方便代理，我们监测在不存在和存在去污剂的情况下β-内酰胺酶的抑制。在去污剂存在下减弱的抑制是基于聚集体的机制的特征。在本文所述的96孔格式测定中，每小时可以一式两份地测试约200个分子的洗涤剂依赖性灵敏度。此外，我们还描述了简单的实验，可以提供额外的确认聚集为基础的抑制。
At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of beta-lactamase in the absence and presence of detergent. Inhibition that is attenuated in the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional confirmation of aggregate-based inhibition.