Detection of ligands from a dynamic combinatorial library by X-ray crystallography
Detection of ligands from a dynamic combinatorial library by X-ray crystallography
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DOI：
10.1002/anie.200351951
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发表时间：
2003-01-01
影响因子：
16.6
通讯作者：
Jhoti, H
中科院分区：
文献类型：
--
作者：
Congreve, MS;Davis, DJ;Jhoti, H
动态组合化学（DCC）是一种分子识别方法，其中组合文库的特定成员被选择并使用模板扩增。[1-6]DCC和传统组合化学之间的主要区别在于，DCC中将构件连接在一起的反应是可逆的，并且在热力学控制下，动态组合库（DCL）的不同成员之间存在持续的交换。[7]因此，DCL能够响应分子识别事件，这是由于模板（例如蛋白质）的存在，其可以稳定文库的特定成员并诱导平衡的移动，从而有利于所选种类的形成。该方法的缺点是，它通常需要过量的蛋白质来观察对平衡的影响。此外，只能使用质谱分析或HPLC色谱图作为指纹，通过比较存在和不存在蛋白质成分的相同库来检测这种效果。我们现在报告一个互补的方法，其中配体直接观察到的X射线晶体学的解释的电子密度图暴露于一个动态的组合库混合物的晶体。我们称这项技术为动态组合X射线晶体学或DCX。这种方法被用来检测快速有效的细胞周期蛋白依赖性激酶2蛋白的抑制剂。DCX与先前报道的DCC技术相比具有关键优势[8，9]，因为可以从DCL中组分的混合物中直接识别配体，并且其详细的结合模式可以从电子密度图中定义。此外，与先前报道的DCC方案相比，每个单独的DCX实验仅需要非常少量的蛋白质。已经用X射线晶体学和NMR光谱技术确定，非常小的分子或“片段”（MW= 100-200）能够以可重现和特异性的方式与蛋白质结合，即使它们的内在效力（用体外生物测定法测定）非常弱（IC 50 μm-mm）。[10-14]我们假设，与蛋白质活性位点内相邻口袋结合的片段原则上可以自组装，以产生更大，更有效的配体，如果它们具有互补的化学反应性。[15]这种方法将具有关键优势，因为可以避免耗时和昂贵的常规合成，纯化和测试设计用于发现有效抑制剂的组合文库的实践。
Dynamic combinatorial chemistry (DCC) is an approach to molecular recognition in which specific members of a combinatorial library are selected and amplified with the use of a template.[1–6] The principle difference between DCC and traditional combinatorial chemistry is that the reaction linking the building blocks together in DCC is reversible and there is an ongoing interchange between the different members of the dynamic combinatorial library (DCL) under thermodynamic control.[7] A DCL is therefore able to respond to molecular recognition events owing to the presence of a template, such as a protein, which can stabilize a particular member of the library and induce a shift in the equilibrium, favoring the formation of the selected species. A drawback of the method is that it usually requires excess quantities of protein for an effect on the equilibrium to be observed. Also, the effect can only be detected by comparison of identical libraries, generated with and without the protein component present, using either mass spectrometric analysis or HPLC chromatograms as a fingerprint. We now report a complimentary approach in which ligands are observed directly by X-ray crystallography by interpretation of electron-density maps from crystals exposed to a dynamic combinatorial library mixture. We call this technology dynamic combinatorial X-ray crystallography or DCX. This approach was used to detect rapidly potent inhibitors of the cyclin-dependent kinase2 protein. DCX has key advantages over previously reported DCC technologies [8, 9] in that direct identification of the ligand is possible from the mixture of components in theDCL, and its detailed binding mode is defined from the electron-density maps. Furthermore, only very small amounts of protein are required for each individual DCX experiment compared with previously reported DCC protocols. It has been established with both X-ray crystallography and NMR spectroscopic techniques that very small molecules or “fragments”(MW= 100–200) are capable of binding to proteins in a reproducible and specific manner, even though their intrinsic potency, as determined with an in vitro biological assay, is very weak (IC50 μm–mm).[10–14] We postulated that fragments bound to adjacent pockets within the active site of a protein could, in principle, self-assemble to generate larger, more potent ligands if they had complimentary chemical reactivities.[15] Such a method would have key advantages, as the time-consuming and expensive practice of conventional synthesis, purification, and testing of combinatorial libraries designed to discover potent inhibitors might be obviated.