NMR spectroscopy in structure-based drug design Roberts 43 mobile parts of the structure, and become more rigid spectrum having single peaks for the backbone amide of when the ligand binds. Thus, in both cyclophilin A[20] most residues in the protein. Provided that the residues from and in the intestinal fatty-acid binding protein which all these peaks arise have been identified, changes in [21,22,23, local regions of the structure are less well the position of these peaks on addition of ligand can quickly defined than the remainder and appear by a number of give an indication of where the ligand binds. This approach criteria to be mobile. On the addition of ligand, these has been adapted for screening for lead compounds(see regions become structurally better-defined and less Moore, this issue, pp 54-58). It has also been applied at a mobile- although in the intestinal fatty-acid binding later stage in the design process to establish whether new homology(PH)and phosphotyrosine binding(PTB) or derived from chemical libraries)do bind in the same way domains [24, 25, 26], some residues involved in ligand as the parent'compound [36, 37 ]. Additional information of binding are already restricted in the unligated protein, the same kind but including backbone carbonyl as well as while others are mobile and become restricted only NH groups can be obtained from the HNCO experiment when the ligand binds [38], and the TROSY experiment [14"]will allow the exten sion of this kind of experiment to much larger proteins In all these examples, the observed mobility involves a These experiments give information only on the protcin very limited number of residues(-5-20) One function of backbone, rather than on the sidechains that may be more this localised mobility in cyclophilin and the fatty-acid directly involved in binding: an approach binding protein may be to allow access of the ligand to a sidechain interactions is selective 13C-labelling of individual rather deep binding pocket. In cases where the residues residue types(e. g. methionines)[39] mobile are directly involved in ligand bindi degree of active-site mobility is required to allow the pro- Nuclear Overhouser effects(NOEs), which provide infor- rein to bind a range of ligands, Fluctuations, perhaps mation on inthernuclear distances, have the advantage correlated, in both ligand and binding site ar s of peptide for the proximity of an atom on the ligand and one on the [27, 28 In these and other cases, a knowledge of this y Stein, and hence a clear identification of the binding site recognition, for example by SH2 and SH3 domains disadvantage of this technique at the experiments mobility could in principle be exploited in the design of required to identify and assign intermolecular NOEs can new structurally diverse ligands be more time consuming. If the ligand binds relatively weakly(Kd210-7M), the transferred NOE approach(see Although the observation of residues in the binding site below )is very useful, whereas for tightly bound ligands a xhibiting relatively large-amplitude motions in the absence number of 'editing experiments ([40,41] and references of ligand and becoming'immobilisedon ligand binding is a therein)make it possible to specifically identify NOes common one, it is far from universal. Increases in mobility of between a 1.C-and/or 15N-labelled protein and an unla some residues on complex formation have been reported belled ligand. If a sufficient number of intermolecular [28, 29, 30]. The entropy changes resulting from these NOEs can be identified, they can be used to'dock'the lig Canges in local mobility[29-31] are in several cases signifi- and into the site; this has been successfully applied to the cant in relation to the overall free energy of binding, and binding of inhibitors to dihydrofolate reductase [42-44] must be considered in a detailed analysis of the individual and stromelysin [45 and a valuable assessment of the contributions to binding. A report of the changes in mobility effectiveness of this method has been reported [461 f the binding site of SH2 domains on binding of phospho- peptides includes a valuable discussion of the relation If the protein of interest contains a paramagnetic centre, between binding-site mobility and binding energy [28""I. such as a metal ion, relaxation effects of the unpaired elec tron(s)of the metal on the nuclei of the ligand can be In addition to molecular motion per se, other dynamic to determine distances from the metal to atoms of the processes which are significant in the context of drug design bound ligand. These distances can then be used to dock can also influence NMR spectra, notably tautomeric and the ligand into the site without the need for protein reso- ionisation equilibria. These can only be inferred from X-ray nance assignment. This has recently been applied to the crystallography but can often be studied much more direct- study of substrate binding to the drug-metabolising ly by NMR, as illustrated tudies of substrate and cytochromes P450 147, 48", 49, 50,51l, offering the prospect nhibitor binding to dihydrofolate reductase [32, 33, 34 of a structurally-based understanding of the specificity of these enzymes for use in drug design Structures are not yet Location of binding sites and docking available for mammalian cytochromes P450: the NMR nMr has proved to be particularly valuable in providing a information has been used either to compare the positions rapid identification of the location of a ligand binding site. An of a number of substrates relative to the haem 51l, or, by experiment widely used in this connection is 15N-'H het- combining the nmR and homology information together to pronuclear single or multiple quantum correlation(HSOC or construct models of individual enzyme-substrate complex HMOC, respectively), which quickly [35] yield a simple es [47, 48], which simultaneously satisfy the constraints of