Fesik S W, Zuiderweg E R, Olejniczak E T, Gampe R T
Pharmaceutical Discovery Division, Abbott Laboratories, IL 60064.
Biochem Pharmacol. 1990 Jul 1;40(1):161-7. doi: 10.1016/0006-2952(90)90191-m.
Four approaches are described for providing detailed structural information on large enzyme/inhibitor complexes to aid in the design of improved enzyme inhibitors. In one approach, proton NMR spectra are simplified by isotope-editing procedures in which only those protons that are attached to isotopically labeled nuclei (e.g. 13C or 15N) and their scalar or dipolar coupled partners are observed. Using this strategy, the conformation of an inhibitor bound to porcine pepsin can be determined and structural information on the active site obtained. In another approach, two-dimensional nuclear Overhauser effect (2D NOE) difference spectra are obtained by subtracting NOE spectra of two enzyme/inhibitor complexes prepared with either a protonated or a deuterated inhibitor. Only NOEs arising from protons of the inhibitor substituted with deuterium appear in the 2D NOE difference spectra as illustrated for a pepsin/inhibitor complex. In a third strategy, deuterated enzymes are employed to eliminate the many proton NMR signals of the enzyme and allow the selective detection of the resonances corresponding to the bound ligand as demonstrated for CTP bound to CMP-3-deoxy D-manno-octulosonic acid (KDO) synthetase. Finally, a fourth approach is described using heteronuclear three-dimensional NMR spectroscopy in which homonuclear 2D NMR spectra are edited with respect to the heteronuclear chemical shifts. Using these methods the complete three-dimensional structures of large enzyme/inhibitor complexes can potentially be obtained. Examples of the spectral simplification that can be achieved using 3D NMR are given for 15N-labeled CMP-KDO synthetase complexed with an inhibitor and CTP.
本文介绍了四种方法，用于提供大型酶/抑制剂复合物的详细结构信息，以辅助设计更优的酶抑制剂。第一种方法是通过同位素编辑程序简化质子核磁共振谱，该程序仅观察那些与同位素标记核（如(^{13}C)或(^{15}N)）相连的质子及其标量或偶极耦合的伙伴质子。运用此策略，可确定与猪胃蛋白酶结合的抑制剂的构象，并获得活性位点的结构信息。第二种方法是通过减去用质子化或氘代抑制剂制备的两种酶/抑制剂复合物的核Overhauser效应（NOE）谱，来获得二维核Overhauser效应（2D NOE）差异谱。如胃蛋白酶/抑制剂复合物所示，只有被氘取代的抑制剂质子产生的NOE出现在2D NOE差异谱中。第三种策略是使用氘代酶来消除酶的许多质子核磁共振信号，并允许选择性检测与结合配体相对应的共振信号，如CTP与CMP - 3 - 脱氧 - D - 甘露糖 - 辛酮糖酸（KDO）合成酶结合时所示。最后，描述了第四种方法，即使用异核三维核磁共振光谱，其中同核二维核磁共振谱根据异核化学位移进行编辑。使用这些方法有可能获得大型酶/抑制剂复合物的完整三维结构。给出了使用三维核磁共振可实现的谱图简化示例，即(^{15}N)标记的CMP - KDO合成酶与抑制剂和CTP形成的复合物。
