Methods of using click chemistry in the discovery of enzyme inhibitors
Methods of using click chemistry in the discovery of enzyme inhibitors
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DOI：
10.1038/nprot.2007.323
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Yao, Shao Q.
中科院分区：
文献类型：
--
作者：
Srinivasan, Rajavel;Li, Junqi;Yao, Shao Q.
该协议描述了一步一步的程序，用于有效地组装的双齿抑制剂库的目标酶，使用所谓的“点击化学”之间的炔轴承核心组和叠氮化物修饰的外围组，然后通过直接的生物筛选的潜在的“命中”的识别。该反应突出了其模块性，高效率（在大多数情况下类似于100%产率）和对片段中存在的许多官能团的耐受性，以及生物相容性（通常在水性条件下使用少量生物相容性催化剂进行）。该方法由三个步骤组成：（i）化学合成含炔的蛋白酪氨酸磷酸酶或基质金属蛋白酶核心基团和不同的叠氮化物修饰的外围基团;（ii）点击化学以组装双齿抑制剂文库;和（iii）使用384孔微孔板测定法直接筛选具有靶酶的文库。在核心和外围基团的化学合成和点击化学条件的优化（类似于1周）之后，步骤（ii）和（iii）花费3天完成（类似于文库组装的1-2天和抑制剂筛选的1天）。
This protocol describes the step-by-step procedures for the efficient assembly of bidentate inhibitor libraries of a target enzyme, using the so-called 'click chemistry' between an alkyne-bearing core group and an azide-modified peripheral group, followed by direct biological screening for the identification of potential 'hits'. The reaction is highlighted by its modularity, high efficiency (similar to 100% yield in most cases) and tolerance toward many functional groups present in the fragments, as well as biocompatibility (typically carried out in aqueous conditions with small amounts of biocompatible catalysts). The approach consists of three steps: (i) chemical synthesis of alkyne-bearing protein tyrosine phosphatase or matrix metalloprotease core groups and diverse azide-modified peripheral groups; (ii) click chemistry to assemble the bidentate inhibitor libraries; and (iii) direct screening of the libraries with target enzymes using 384-well microplate assays. Following the chemical synthesis of the core and peripheral groups and optimization of the click chemistry conditions (similar to 1 week), steps (ii) and (iii) take 3 d to complete (similar to 1-2 d for library assembly and 1 d for inhibitor screening).