NMR methods for determining the structures of enzyme/inhibitor complexes as an aid in drug design.

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.