NMR spectroscopy in structure-based drug design Gordon cK roberts NMR methods for the study of motion in proteins continue to structure and dynamics of the binding site improve, and a number of studies of protein-ligand complexes Methodological developments in NMR of macromolecules relevant to drug design have been reported over the past year, continue apace [10, 11]. Important recent developments for example, studies of fatty-acid-binding protein and SH2 and che use of residual dipolar couplings and of SH3 domains. These studies have begun to give a picture of anisotropic contributions to relaxation to provide new the structural dynamics of protein-ligand complexes and to kinds of structural information [12, 13]; measurements of relate the changes in dynamics on ligand binding to the origins dipolar couplings in partially oriented proteins are a partic of specificity. NMR is also valuable in locating binding sites, ularly important addition to NMR methods for structure both qualitatively from changes in chemical shift and more determination, as they provide information on the orienta- precisely from distances measured from relaxation effects. The tion of individual bond vectors within the molecule conformation of the bound ligand can provide useful information Another exciting development is the exploitation of rela for drug design, and over the past year improvements in ects to obtain methods have made it easier to obtain quantitative information proteins -up to -150 kDa[14]. The ability of nmr to from transferred nuclear Overhauser effect experiments provide information on dynamic processes has long been recognised, and developments in NMR studies of molecu Addresses lar motion in proteins have been reviewed [15I Road, Leicester LE1 9HN, UK; e-mail: gcr@le ac uk In thinking about structure-based drug design, molecular motion in the binding site is of fundamental importance for Current Opinion in Biotechnology 1999, 10: 42-47 the access of the ligand and the kinetics of association and dissociation, and in many cases for determining the struc tural range of ligands that can bind. In this context, motions C Elsevier Science Ltd ISSN 0958-1665 in the microsecond-millisecond range are probably more important than those in the nanosecond range [16], because E nuclear Overhauser effect this is the ale in which ligand binding and dissociation take place. In structures determined by NMR, there are invariably regions that are 'less well determined than oth ntroduction ers-which differ more than the average between As recently noted in this journal by Amzel [1], it seems members of the ensemble of structures. A recent analysis kely that combinatorial chemistry and structure-based provides evidence that ill-definedregions in the methods will cease to be rival approaches to drug design NMR-derived ensemble correspond to regions une and instead will be combined to maximise the effective- relatively large-amplitude low-frequency motions zL an ness of the design process [1, 2. NMR spectroscopy has in practice the regions of the structure that are poorly deter a Valuable role to play in both approaches, and a number mined often also show relaxation behaviour characteristic of significant recent advances in its application are internal motion, The motion responsible for relaxation reviewed in this issue(see Watts, pp 48-53; Moore, effects may, however, be on a very different,shorter pp 54-58)and elsewhere [3] timescale from that responsible for the apparent'disorder' which leads to the observation of"ill defined regions in the In recent years, NMR has contributed in a major way to structure. Although relaxation experiments can qualitatively protein and nucleic acid structure determination, with demonstrate nanosecond mobility without ambiguity over 100 new structures reported annually. NMR can detailed description of its nature and frequency is more thus, like crystallography, provide the 'raw material' for ficult[15]. Rotating frame relaxation measurements afford structure-based drug design -the structure of the target a direct approach for measuring dynamics on the microsec molecule and of its complexes with candidate drugs. ond-millisecond timescale [15, 18), whereas in favour Recent examples of NMR structures of drug-target com- cases, lineshape analysis allows both rate measurement and plexes include complexes of dihydrofolate reductase [4] characterisation of the nature of the motion. For example, and HIV protease [5, 6, with inhibitors, and complexes experiments of this kind have led to the interesting conclu of antibiotics with oligonucleotides [7, 8"]. The focus sion that an arginine guanidino group and a ligand of this review will, however, be on recent developments in two other aspects of NMR in drug design: first, going and dihydrofolate reductase undergo correlated motion(191 beyond the static (or average) structure to characterise so that the ion-pair is maintained as the structure fluctuates the binding site flexibility; and second, obtaining infor- mation short of a full struct n the location of the Several examples have been reported of proteins in binding site and the conformation of the ligand within it. which parts of the ligand binding site are the