44 Analytical biotechnology he NMr-determined distances and of the homology with Conclusion bacterial cvtochrome P450s of known structure The diversity of applications of NMR to structure-based drug design parallels the range of its applications to protein struc- Determination of the conformations of ture in general, and both have seen significant advances over the past year. NMR is particularly useful in characterising In the absence of detailed information on the structure of the motion in proteins; understanding binding site flexibility will drug-target complex, a knowledge of the conformation of be important in designing novel ligands and, at a fundamer the bound drug for the natural ligand one wishes to mimic) tal level, in accounting for entropic contributions to affinity at can provide a valuable constraint on the drug design process d of indivi The transferred NOE complex is not available, the fact that Nmr can be used to in this regard (52-55); although limited to ligands with study a number of compounds relatively rapidly gives it an Kd210-7M, it requires relatively little protein and can be advantage in identifying the binding site for candidate ligands and determining their conformation in the bound describes its use to study the conformation of a macrolide Several recent methodological advances discussed here antibiotic bound to the ribosome 156. Recent advances in promise to improve the precision of the information obtained the quantitative analysis of the experiment have made pre. and to extend the range of applicability of the methods, lead- cise structures of the bound ligand easier to obtain. The two g to an expansion in the use of NMR as a valuable key steps in the quantitative analysis are to account for any component in the armoury of the drug designer contributions from the exchange process and to ensure that the NOEs between ligand protons that yield distance con- References and recommended reading of review straints are direct effects and not mediated by magnetisation Papers of particular interest, published within the annual perid transfer via other protons of the ligand or the protein(spin diffusion ). Identification of spin diffusion can be helped by using the rotating frame NOE experiment [57, 58), in which he indirect NOE contributions have opposite sign in con 1. Amzel LM: Structure-based drug design. Curr Opin Biotechno 1998,9:366-369 junction with the normal NOE experiment. Spin diffusion 2. integration of structure-based drug design and combi through ligand protons and exchange contributions can accounted for by using a combined relaxation and exchange chemistry for efficient drug discovery. Structure 1997,5: 319-324 matrix in the data analysis [58, 591, but spin diffusion through 3. Stockman BJ: NME the protein presents more of a problem. The most general A drug design solution is to remove the problem by per-deuteration of the tic dy -triet syste ms, incudis a nm thein aplin inv protease. r of sre- have been described for quenching spin diffusion by use of pulse to select both thesourceand'target'nuclei and thus obtain distance constraints free of the effects of spin diffu- 5. Wlodawer A, Vondrasek Inhibitors of Hiv-1 protease: a major sion: its application to NAD+ bound to lactate dehydrogenase [63] illustrates its promise in studies of An informative and critical review of the contributions of crystallography and 6. Yamazaki T, Hinck Y, Nicholson LK, Torchia DA, Wingfield Illustrative examples of recent analyses of the conformation of bound ligands by analysis of transferred NOEs include a novel cyclic urea-type inhibito etermined by nuclear magnetic resonance Protein Sci1996 studies of enzyme-substrate/inhibitor 158, 63, 64, 65]. 5:495-506 protein-carbohydrate 166,67,68""] and protein-peptide 7. Chen H, Liu X, Patel DJ: DNA bending and unwinding associated [69,70, 71,72.interactions, Among the interesting points actinomycin D antibiotics bound to partially overlapping sites om this work are evidence in several sy on DNA. J Mol Biol 1996. 258: 457-479 that two-step kinetic mechanisms(E+LAEL 6EL 8. Kumar RA, Ikemoto N, Patel DJ: Solution structure of the esperamicin A1-DNA complex. J Mol Bio/1997, 265: 173-186 where E is enzyme and L is the ligand)can have important See annotation to [g] effects on the experiment, under some circumstances the 9. Kumar RA, Ikemoto N, Patel DJ: Solution structure of conformation obtained being that in the intermediate(el) This and the prentitumg r apei B] d rather than the final complex [58, 59, 65), and evidence for the struc ligands bound in a high-energy conformation [66]. There structures provide explanation ved sequencespecificity of the are also examples where peptides corresponding to key positions the proradical centres inding loops of a protein have a different conformation oproprately for double-strand when bound to a target protein than in the free parent' pro- 0. Clore GM, Gronenborn AM: New methods of structure refinement rein [72,73]; this has obvious implications for the design of peptidomimetic inhibitors by NMR, inciwiew of some recent developments in ture determination ding the application of the methods in [12, 13