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วิจัยต้นอ้อ

  1. 1. 285 NMR, already some 50 years old, has long been an invaluable analytical method in industry for verification of chemical synthesis and compound characterisation. The range of molecular information accessible through NMR, however, offers a far larger horizon of applications. Of these, ligand screening by NMR has emerged as a very promising new method in drug discovery. Its unmatched screening sensitivity, combined with the abundance of available information on the structure and nature of molecular binding, justifies the growing interest in this dynamically expanding NMR application. Addresses *NOVASPIN Biotech GmbH, Mühlfeldweg 46, 85748 Garching, Germany; e-mail: info@novaspin.de †Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstraße 4, 85747 Garching, Germany; e-mail : kessler@ch.tum.de Current Opinion in Chemical Biology 2001, 5:285–291 1367-5931/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations HPLC high-performance liquid chromatography MAS magic-angle spinning NOE nuclear Overhauser enhancement NOESY NOE spectroscopy RDC residual dipolar couplings S3 spin-state selection STD saturation transfer difference TOCSY total correlation spectroscopy trNOE transferred intramolecular NOE TROSY transverse relaxation-optimized spectroscopy Introduction NMR has gained widespread acceptance over recent years as a most versatile tool for industrial drug research. The relevance of NMR in this regard rests on two pillars — its use as a powerful, universal and fast-screening technique to detect intermolecular interactions with unparalleled sensitivity, and its potential to yield unique information with atomic resolution to guide structure-based drug design. This review reflects this dual importance, focussing not only on NMR screening techniques, but also including structural applications and a brief survey of NMR hardware and general methodological developments that are contributing to advances in the field. The reader is also referred to other reviews [1–5]. Basic developments in NMR The application of NMR to industrial drug research is greatly facilitated by recent hardware developments (reviewed in [6]). Most important for screening purposes are developments that reduce sample amounts and dead- times for sample handling — notorious shortcomings of ‘wasteful’ NMR. Cryoprobe technology can substantially increase the signal-to-noise ratio, which also translates into lower accessible binding affinities. Flow probes alleviate the need for NMR tubes and their time-consuming handling [7••], enabling direct coupling with separation techniques such as (HP)LC. Microcoil probes reduce the required sample volume and also offer superior radio-frequency field homogeneity [8], benefiting difference-based NMR screening methods. Screening techniques are also beginning to benefit from recent fundamental advances in NMR that have already proven of value in structure determination. These include spin-state selection (S3), which is most often employed to optimise relaxation properties (i.e. in transverse relaxation- optimized spectroscopy; TROSY [9]), extending molecular sizes amenable to NMR well beyond 100 kDa, primarily by enhancing spectral resolution. S3 experiments are also employed [10] to precisely measure residual dipolar couplings (RDC) [11], a new set of unique structural constraints that have proven useful in orienting (hydrogen-poor) ligands bound to macromolecular targets. RDC result from partial molecular alignment that can be brought about by adding magnetically orienting media such as bicelles, filamentous phages, and others [12]. Connectivities across hydrogen bonds [13] also yield new structural constraints that can com- plement the scarce nuclear Overhauser enhancement (NOE) data in hydrogen-poor systems. Last but not least, various new strategies for isotopic labelling of proteins, crucial to NMR, have been developed (for a recent review, see [14]). General aspects of NMR screening NMR offers several key advantages for drug research: 1. NMR can detect the weakest ligand–target interactions of any method so far and thus discover initial hits with even millimolar binding constants. 2. NMR is a universal screening technique that, unlike bioassays, requires no knowledge of a protein’s function and therefore no target-specific set-up. Thus, protein targets that have been identified purely on a genomics basis and so far lack a functional bioassay can immediately be screened by NMR. 3. NMR concomitantly enables a determination of binding constants [15]. 4. NMR spectrally separates individual components, allowing the direct screening and deconvolution of mixtures from natural sources or combinatorial chemistry. 5. In addition to the mere binary binding information, NMR returns crucial structural information for both target and ligand with atomic resolution for the subsequent optimisation of weak initial hits into strongly binding drug candidates. Applications of NMR in drug discovery Tammo Diercks*, Murray Coles* and Horst Kessler†
  2. 2. NMR screening exploits the fact that many NMR observ- ables change even upon temporary binding because of global and local effects (see Figures 1 and 2). The effects of binding may be monitored either on the target or on the ligand and are summarised in Table 1. The observed molecule primarily dictates inherent limitations such as sample availability (possibly with appropriate isotope labelling), solubility, stability and molecular size. Ligand-observed screening Detection with these techniques generally takes place on the dissociate, free form of the small ligand (implying a minimal Kd of ca. 10–7 M), allowing the direct identification of bind- ing species from mixtures. The unobserved target molecule is not subject to size limitations and may even be immo- bilised. In general, isotopic labelling is not required. Many of these techniques rely on size-dependent effects, which can be difficult to resolve for weak binding (implying a maximal Kd of ca. 10–3 M) and small relative molecular size differ- ences between ligand and target. These effects can be selected for in 1D filtered spectra, in which tuning the filter selects for different ligand affinities. Alternatively, the size of the effect could be evolved in a second indirect dimension, yielding pseudo-2D edited spectra. The information for structure-based ligand optimisation obtainable through these techniques is more limited than for target-observed tech- niques. It may, however, reveal a ligand’s binding epitope, its bound conformation, and even contacts to other ligands binding at adjacent sites. Use of molecular diffusion Detection of transiently binding ligands by their decreased diffusion coefficients is an established screening technique (for exhaustive reviews see [16,17•]) Pseudo-2D diffusion- edited experiments (diffusion-ordered spectroscopy, DOSY), where molecules are separated according to their individual diffusion coefficients, are particularly useful for deconvoluting mixtures. Alternatively, 1D diffusion filters can be combined with standard 2D correlation experi- ments, such as in the diffusion-modulated gradient COSY (correlation spectroscopy) [18] and diffusion-encoded total correlation spectroscopy (TOCSY; DECODES) [19], to elucidate spin systems and facilitate mixture analysis. Diffusion filtering or editing might also be enhanced by combination with other screening techniques, such as NOE pumping [20,21] (see below). In a promising appli- cation for combinatorial chemistry, diffusion filtering has recently been used in solid-state MAS (magic-angle spinning) NMR to identify and distinguish resin-bound molecules from impurities [22]. Use of relaxation Screening based on binding-induced slowing of global dynamics, leading to a general decrease in NMR relaxation times, is conceptually very similar to diffusion-based screening. The diffusion filter is replaced by an inversion- recovery delay, a spin-lock period or a CPMG echo to filter for the different types of relaxation times, T1, T1ρ and T2, respectively. One-dimensional relaxation-based screening usually requires subtraction of a reference spectrum of the target-free ligand mixture, and measures must be taken to avoid artefacts due to binding-induced lineshape distor- tions, chemical-shift changes and residual target signals [23]. T2-based ligand screening may also be performed by comparing spectral linewidths [24•] that increase upon binding. Complications can arise from superposition of line-broadening due to chemical exchange or slow 286 Combinatorial chemistry Figure 1 Global NMR consequences of ligand–target interactions. Observation is restricted to the ligand where the effects are most pronounced. Their strength depends on molecular size differences and binding kinetics. kA kD + Complex properties similar to target properties • Slow Relaxation • Fast diffusion • Positive NOE • Fast relaxation • Slow diffusion • Negative NOE Small ligands ComplexLarge target Current Opinion in Chemical Biology Figure 2 Local NMR effects of complex formation may be observed on the ligand and/or the target. Structural information is available as effects are mostly localised to the binding site. Surface protection Current Opinion in Chemical Biology Intermolecular magnetisation transfer Chemical shift perturbation Perturbation of local dynamics{
  3. 3. conformational motions (on the microsecond and millisecond timescales), which are often relevant for induced-fit during intermolecular interactions and damped upon binding. Thus, a size-dependent decrease in T2 times might be compensated for by a flexibility-dependent reduction in line-broadening. In contrast, the recently proposed concept of transferred T1 times [25] relies on the fact that relaxation rates measured on a fast-exchanging ligand in the free state also contain contributions from its local dynamics in the bound state, as immobilisation of flexible regions of the ligand generally increases relaxation rates. Transferred relaxation rates may therefore be seen as a dynamic analogue of transferred NOE (see below). Individual differences in relaxation rates must be compared with their average in order to distinguish local from global dynamic changes [25]. Another complication arises from local relaxation contributions because of molecular anisotropy. Intramolecular magnetisation transfer: transfer-NOE Transferred intramolecular NOEs (trNOEs) are well established as unique sources of structural information on the bound ligand (for a review see [26]) where spin-diffu- sion should be suppressed to obtain optimal structural precision [27,28]). The observation of trNOE has proved to be equally useful in the fast screening of ligand mixtures [24•], forming the basis of ‘bioaffinity NMR’ [29]. The technique relies on the size-dependence of the intramolecular NOE, which shows slow NOE build-up with weak positive maxima for free ligands and rapid build-up with strong negative maxima for the bound state. If dissociation of the ligand occurs quickly enough (i.e. Kd > 10–7 M), a sufficient percentage of observable free ligand will retain intense negative trNOE as a ‘memory’ of the bound state and thus quickly indicate binding. If the residence time is too short (i.e. Kd > 10–3 M), however, trNOE will build up too weakly for detection [30]. Routinely, trNOE are recorded as 2D NOESY (NOE spectroscopy) spectra with short mixing times favouring build-up of trNOE over direct NOE. Relaxation filtering suppresses residual target signals. Notable are applica- tions of trNOE-based screening [30,31•,32] and recent experimental improvements [33]. Screening by trNOE imposes upper limits on the ligands (< 1 kDa), which have to fall into the positive NOE regime. For borderline cases, this regime may be extend- ed, for example, by increasing the temperature [31•]. Another problem is the possible cancellation of positive direct and negative transferred NOE, which could be resolved by more lengthy 3D experiments such as the 3D TOCSY–trNOESY [34], or by resorting to other tech- niques such as saturation transfer difference (see below). Intermolecular magnetisation transfer The intermolecular magnetisation transfer is always posi- tive and cancellation cannot occur. For its measurement, non-equilibrium magnetisation must first be created asym- metrically (i.e. on one component only, usually the drug target). It is then transferred to the binding partner during a mixing period. The intermolecular magnetisation trans- fer results in altered signal intensities for both components and can finally be monitored on either the starting or the receiving molecule (usually the ligand). Remaining target resonances can be suppressed (e.g. by isotope filtering or relaxation filtering). Subtraction of a reference spectrum (recorded without initially disturbing the magnetisation) is usually required to reveal these intensity changes and entails the risk of subtraction artefacts. Asymmetric non-equilibrium magnetisation on the target molecule may generally be created by selective irradiation, requiring the existence of a unique NMR frequency band. Applications of NMR in drug discovery Diercks, Coles and Kessler 287 Table 1 NMR observable effects of ligand binding. Global effects of binding Ligand observed Screening Applications References Translational dynamics Diffusion rates Affinity NMR [17•,19] Rotational dynamics Relaxation rates Line broadening [23,24•] Sign of transferred intramol. NOE Bioaffinity NMR (trNOE) [29,30,31•,32,34] Molecular orientation Residual dipolar couplings * Local effects of binding Ligand- or target-observed Chemical shift perturbation Chemical shifts SAR-by-NMR (HSQC screening) [44•,48,49,51,53•] Local dynamics perturbation Relaxation rates * Surface protection Water exchange rates * Paramagnetic surface probing * Intermolecular magnetisation transfer Via scalar couplings * (e.g. intermol. H-bonds) Via nuclear dipolar couplings Bioaffinity NMR (STD) [32,35,37•] (intermol. NOE) (Reverse) NOE pumping [20,21] WaterLOGSY [38] Via electron dipolar couplings (paramagnetic relaxation) SLAPSTIC [39•,41•] *No application to NMR screening reported yet. Intermol., intermolecular.
  4. 4. Initially localised, it is spread across the entire molecule by spin diffusion such that magnetisation may be picked up by a ligand binding anywhere on the surface. Alternatively, non-equilibrium magnetisation may be created symmetri- cally, followed by asymmetric suppression making use of the large size difference between target and ligands. For instance, a diffusion filter would suppress magnetisation on the ligands, whereas a relaxation filter would suppress magnetisation on the target. Saturation transfer difference (STD) [35] as the special case of intermolecular steady-state NOE is observed after frequency-selective irradiation of the target and subtrac- tion of a reference spectrum with off-resonance irradiation. Although usually run as 1D spectra, STD can be coupled to standard 2D correlation experiments such as TOCSY, COSY and HMQC (heteronuclear multiple quantum correlation) to alleviate spectral overlap [32,35]. The distance-dependence of the observed signal intensities identifies those nuclei closest to the target–ligand interface. This ‘epitope mapping’ [28,36] may be seen as a ligand-based complement to the ‘SAR-by-NMR’ approach. A remarkable application extends STD screening to solid-state NMR [37•], in which the target was immobilized on controlled-pearl glass, facilitating its recovery. From a comparison of STD integrals, it was possible to assess relative binding strengths of the dissolved ligands. A reference spectrum with pure target-free resin must rule out direct ligand–resin interactions. NOE pumping [20] employs the scheme of symmetric magnetisation inversion, followed by asymmetric suppres- sion. Classically, the ligand magnetisation is suppressed via diffusion filtering, and its recovery directly indicates inter- molecular magnetisation transfer from the target, hence binding. A suppression of remaining target magnetisation prior to acquisition greatly facilitates identification, but the diffusion filter employed may entail serious intensity losses through concomitant relaxation of the target magnetisation. This problem is alleviated in the reverse case (reverse NOE pumping) [21], in which ligand magnetisation is preserved while target magnetisation is suppressed via relaxation or isotope filtering. The ligand magnetisation is then reduced because of transfer onto the target, and signal reductions observed on a ligand thus indicate binding. These reductions, however, are only revealed after subtrac- tion of a reference spectrum, which also takes account of unspecific magnetisation losses through T1 relaxation. WaterLOGSY [38] exploits the surface layer of target- bound water molecules as a pool of selectively invertible magnetisation that can be transferred onto binding ligands. After simultaneous selective water suppression and relaxation filtering (for target suppression), only signals of binding ligands are detected. Paramagnetic spin labels Magnetic spin interactions can be enhanced drastically by introducing paramagnetic spin labels. The induced NMR effects — resonance shifts and line broadening — can thus be observed over larger distances (up to some 20 Å), shorter contact times, and at lower protein concentrations. Soluble paramagnetic reagents have classically been employed to search for metal ion binding sites and probe solvent-accessible surfaces. Intermolecular binding, which blocks part of the solvent-accessible surface, may be detected and localised in a similar manner. In a recent application to second-site screening, a confirmed binding ligand was spin labelled and used to search for ligands concomitantly binding to the target protein at an adjacent site [39•]. The method is analogous to the observation of transferred inter-ligand NOE contacts [40], yet offers larger specificity and sensitivity, establishing connectivity between more distant binding sites. The need to synthesize spin-labelled ligands without affecting their natural binding properties is, however, a major drawback. A blind test must furthermore rule out that ligands interact in the absence of the target. In the SLAPSTIC method, the target protein is spin- labelled to assist in identifying binding ligands [41•]. The line-broadening of ligand signals upon binding is thus strongly emphasized and allows for a reduction in protein (and ligand) amounts by one or two orders of magnitude. It is critical to spin-label the target near, but not within the binding site, as this would invariably lead to altered binding properties. Moreover, no structural information can be derived from this method. Use of residual dipolar couplings Alignment of the target molecule also causes alignment of the ligand upon temporary binding. RDC may thus be transferred from the bound to the isotropic free state if dissociation is fast enough (Kd in the millimolar range) [42,43]. In contrast to NOE data, the structural information available from RDC does not suffer from spin-diffusion and is therefore precise even for large, immobilised targets. RDC yield information on both the bound ligand’s structure and its orientation relative to the target without the need for assigning target resonances as demonstrated in two recent applications [42,43]. This technique has not, however, been applied to ligand screening yet. Target-observed screening Observation on the target, rather than on dissociated ligands, is neither restricted by the ligands’ size nor by an upper affinity limit. It also immediately reveals different binding sites, enables direct distinction between specific and unspecific binding and provides a wealth of structural information for ligand optimisation. This information, however, can only be extracted if the spectral assignments for the target are known. Inherent limitations often preclude observation on the large target molecule, although relaxation-optimised techniques (TROSY) may be of benefit. The amounts of protein required, which must be isotope-labelled, can be reduced significantly by employing 288 Combinatorial chemistry
  5. 5. cryoprobe technology and screening larger mixtures of compounds [44•]. Mixtures of ligands can not, however, be directly deconvoluted. Chemical-shift perturbations Most target-observed screening applications rely on chemical-shift changes as indicators for intermolecular binding. After assignment, these changes can be mapped upon the protein structure, revealing its binding sites [45,46] and guiding structure-based ligand optimisation. This forms the basis of the ‘SAR-by-NMR’ approach [47,48], in which optimised ligands are constructed by linking weakly binding fragments. The approach contin- ues to be successfully used in drug development [49–52]. Standard screening using 2D 15N,1H correlation spectra has recently been complemented by 13C,1H HSQC-based screening on proteins bearing 13C-labeled methyl groups [53•]. The selective labelling, using inexpensive starting materials, avoids the detrimental effects of 13C–13C homonuclear coupling while greatly reducing the cost of sample preparation. This application benefits from the higher proton multiplicity and slower transverse relaxation of the methyl groups. Consequently, intensities were reported to be on average three-times higher. On the other hand, screening based on methyl groups suffers from assignment difficulties and lower spectral dispersion. Note that the distribution and accessibility of (polar) amide and (non-polar) methyl groups are not equivalent, possibly leading to different screening sensitivities. Equating chemical-shift perturbations with spatial proximity to the ligand may be misleading, especially in the presence of large shielding anisotropies in a ligand or because of con- formational changes in the target. Therefore, an alternative has been proposed in which the binding site and the bound ligand’s orientation can be determined more reliably [54] without the need for a complete re-determination of the complex structures. It is based on the comparison of chemi- cal-shift changes in the proximity of the binding site induced by a series of closely related ligands. This can be seen as trading synthetic for spectroscopic efforts. Intermolecular magnetisation transfer The general scheme for intermolecular magnetisation transfer experiments (see above) can also be employed for target-detected screening. In one concept [55], target mag- netisation is selectively suppressed by relaxation filtering after symmetric excitation and then recovers by intermolec- ular transfer from binding ligands. Prior to observation on the target, the ligand signals are filtered out by selective multiple frequency suppression. This, however, precludes the direct screening of ligands with more complex 1H spectra. Screening for macromolecular interactions Screening for macromolecular interactions is a special case in which the ‘ligand’ and ‘target’ have comparable molecu- lar sizes, and size discrimination is impracticable. Other than that, many of the techniques mentioned above can be and have been applied to the problem. For instance, STD was used to probe protein–nucleic-acid interactions by saturating well-separated protons of the latter [56]. A varia- tion of the scheme scans for protein–protein interactions [57] with molecular distinction made by perdeuterating and 15N-labelling only one partner. The unlabelled protein remains susceptible to saturation of the aliphatic protons, while intermolecular STD is observed on the 1H,15N moieties of the labelled protein. The low proton density in this perdeuterated species precludes efficient spin-diffu- sion and the STD is largely confined to the binding site. Design of NMR screening libraries In contrast to other screening techniques, NMR screening requires relatively high sample concentrations and sampling times. With its unprecedented sensitivity (detecting interac- tions with Kd up to 10–2 M) and atomic resolution, however, NMR is particularly suited for guiding the ‘bottom-up’ construction of complex ligands from small, weakly binding molecular scaffolds. In line with this, the principles for com- posing a dedicated ligand library are availability, good solubility, low molecular mass (i.e. < 1–2 kDa), and limita- tion to some 102–103 compounds of maximal diversity. For mixture screening, batches should contain (non-reacting) ligands with isolated NMR signals for easy monitoring. Various attempts have been made to construct libraries using consensus fragments identified from statistical analyses of known drugs (e.g. see [24•,58,59]) and protein-binding ligands [60], and from fragmenting an existing lead compound [52]. Pre-selection of small ligands for NMR screening can also be based on the scoring results obtained from virtual docking [61]. Conclusions NMR screening is now being recognised by industry as a powerful technique for lead discovery. The underlying prin- ciples have mostly been discovered and developed long ago by academic scientists, such that the novelty of NMR screening lies mostly with its applications. In this review, we have offered a classification of the available NMR methods and outlined possible future developments by a reduction to basic NMR properties and principles. The distinction between ligand- and target-observed screening techniques is most important. In practice, a dual strategy that combines the complementary strengths and detectable affinity ranges of both techniques should prove advantageous. Most impor- tantly, ligand-observed techniques may produce false negatives in cases of very strong binding where exchange between target-bound and detected free ligand is too slow. These techniques nevertheless have clear advantages for pre-screening large libraries as they implicitly deconvolute ligand mixtures, require no isotope labelling and pose few limitations on the target. Consecutive target-observed ‘fine- ’screening of the identified binding ligands can then rule out unspecific binding, identify different binding sites and modes, and guide structure-based ligand optimisation. By Applications of NMR in drug discovery Diercks, Coles and Kessler 289
  6. 6. screening a carefully composed small library of basic molecular scaffolds, the pre-screening step may be omitted for amenable targets. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest ••of outstanding interest 1. Moore JM: NMR screening in drug discovery. Curr Opin Biotechnol 1999, 10:54-58. 2. Moore JM: NMR techniques for characterization of ligand binding: utility for lead generation and optimization in drug discovery. Biopolymers 1999, 51:221-243. 3. Roberts GCK: NMR spectroscopy in structure-based drug design. Curr Opin Biotechnol 1999, 10:42-47. 4. Roberts GCK: Applications of NMR in drug discovery. Drug Discov Today 2000, 5:230-240. 5. Stockman BJ: NMR spectroscopy as a tool for structure-based drug design. Progr NMR Spectrosc 1998, 33:109-151. 6. Keifer PA: NMR tools for biotechnology. Curr Opin Biotechnol 1999, 10:34-41. 7. Ross A, Schlotterbeck G, Klaus W, Senn H: Automation of NMR •• measurements and data evaluation for systematically screening interactions of small molecules with target proteins. J Biomol NMR 2000, 16:139-146. This reference gives valuable practical advice on performing industrial-scale ligand screening by NMR, presenting a fully integrated hardware set-up with robotized sample preparation, flow-through probehead and automated data analysis of the acquired 15N,1H-HSQC. A pre-check of the protein–ligand mixtures for aggregation and solubility is required to prevent clogging of the flow-through probe. The authors conclude that screening individual ligands, rather than mixtures, justifies the extra time by alleviating the need for char- acterizing possible ligand–ligand interactions and deconvoluting mixtures. The problem of using too low buffer concentrations, entailing artefactual chemical shift changes, is also discussed. 8. Haner RL, Llanos W, Mueller L: Small volume flow probe for automated direct-injection NMR analysis: design and performance. J Mag Res 2000, 143:69-78. 9. Pervushin K, Riek R, Wider G, Wüthrich K: Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 1997, 94:12366-12371. 10. Permi P, Annila A: Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings. J Biomol NMR 2000, 16:221-227. 11. Prestegard JH: New techniques in structural NMR — anisotropic interactions. Nat Struct Biol 1998, (NMR suppl):517-522. 12. Rückert M, Otting G: Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. J Am Chem Soc 2000, 122:7793-7797. 13. Cordier F, Grzesiek S: Direct observation of hydrogen bonds in proteins by interresidue h3JNC’ scalar couplings. J Am Chem Soc 1999, 121:1601-1602. 14. Goto NK, Kay LE: New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr Opin Struct Biol 2000, 10:585-592. 15. Fielding L: Determination of association constants (Ka) from solution NMR data. Tetrahedron 2000, 56:6151-6170. 16. Johnson CS: Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Progr NMR Spectrosc 1999, 34:203-256. 17. Chen A, Shapiro MJ: Affinity NMR. Anal Chem 1999, • 71:669A-675A. A review describing NMR screening, in particular diffusion-based affinity NMR, in a modular way. 18. Gmeiner WH, Hudalla CJ, Soto AM, Marky L: Binding of ethidium to DNA measured using a 2D diffusion-modulated gradient COSY NMR experiment. FEBS Lett 2000, 465:148-152. 19. Bleicher K, Lin M, Shapiro MJ, Wareing JR: Diffusion edited NMR: screening compound mixtures by affinity NMR to detect binding ligands to vancomycin. J Org Chem 1998, 63:8486-8490. 20. Chen A, Shapiro MJ: NOE pumping: a novel NMR technique for identification of compounds with binding affinity to macromolecules. J Am Chem Soc 1998, 120:10258-10259. 21. Chen A, Shapiro MJ: NOE pumping. 2. A high-throughput method to determine compounds with binding affinity to macromolecules by NMR. J Am Chem Soc 2000, 122:414-415. 22. Shapiro MJ, Chin J, Chen A, Wareing JR, Tang Q, Tommasi RA, Marepalli HR: Covalent or trapped? PFG diffusion MAS NMR for combinatorial chemistry. Tetrahedron Lett 1999, 40:6141-6143. 23. Hajduk PJ, Olejniczak ET, Fesik SW: One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules. J Am Chem Soc 1997, 119:12257-12261. 24. Fejzo J, Lepre CA, Peng JW, Bemis GW, Ajay, Murcko MA, Moore JM: • The SHAPES strategy: An NMR-based approach for lead generation in drug discovery. Chem Biol 1999, 6:755-769. Although no new techniques are described, a clear overview on the set-up of NMR screening, including library design is provided. 25. LaPlante SR, Aubry N, Deziel R, Ni F, Xu P: Transferred 13C T1 relaxation at natural isotopic abundance: a practical method for determining site-specific changes in ligand flexibility upon binding to a macromolecule. J Am Chem Soc 2000, 122:12530-12535. 26. Ni F: Recent developments in transferred NOE methods. Progr NMR Spectrosc 1994, 26:517-606. 27. Vincent SJF, Zwahlen C, Post CB, Burgner JW, Bodenhausen G: The conformation of NAD++ bound to lactate dehydrogenase determined by nuclear magnetic resonance with suppression of spin-diffusion. Proc Natl Acad Sci USA 1997, 94:4383-4388. 28. Maaheimo H, Kosma P, Brade L, Brade H, Peters T: Mapping the binding of synthetic disaccharides representing epitopes of chlamydial lipopolysaccharide to antibodies with NMR. Biochemistry 2000, 39:12778-12788. 29. Meyer B, Weimar T, Peters T: Screening mixtures for biological activity by NMR. Eur J Biochem 1997, 246:705-709. 30. Mayer M, Meyer M: Mapping the active site of angiotensin- converting enzyme by transferred NOE spectroscopy. J Med Chem 2000, 43:2093-2099. 31. Henrichsen D, Ernst B, Magnani JL, Wang W-T, Meyer B, Peters T: • Bioaffinity NMR spectroscopy: identification of an E-selectin antagonist in a substance mixture by transfer NOE. Angew Chem Int Ed Engl 1999, 38:98-102. This article describes an application of trNOE-based screening to a mixture of ligands, also discussing important details to be considered with this tech- nique. Prior to screening, the library was tested for the positive NOE regime, which was eventually reached only after increasing the temperature. The binding ligand then identified by negative trNOE displayed strong spin- diffusion, which in this case helped its identification, but generally blurs structural information. Suppression of residual target protein signals by means of a relaxation filter was largely complete, except for signals from a highly flexible, attached carbohydrate chain. 32. Vogtherr M, Peters T: Application of NMR based binding assays to identify key hydroxy groups for intermolecular recognition. J Am Chem Soc 2000, 122:6093-6099. 33. Weimar T: Improving bioaffinity NMR spectra by means of zero- quantum dephasing. Magn Reson Chem 2000, 38:315-318. 34. Herfurth L, Weimar T, Peters T: Application of 3D-TOCSY-tr-NOESY for the assignment of bioactive ligands from mixtures. Angew Chem Int Ed Engl 2000, 39:2097-2099. 35. Mayer M, Meyer B: Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed Engl 1999, 38:1784-1788. 36. Mayer M, Meyer B: Group epitope mapping (GEM) to identify segments of a ligand in direct contact with a protein: STD NMR as a tool to characterize binding to Ricinus communis agglutinin. J Am Chem Soc 2001, in press. 290 Combinatorial chemistry
  7. 7. 37. Klein J, Meinecke R, Mayer M, Meyer B: Detecting binding affinity to • immobilized receptor proteins in compound libraries by HR-MAS STD NMR. J Am Chem Soc 1999, 121:5336-5337. This article describes an exciting extension of the STD screening method to high-resolution MAS NMR, using dissolved ligands and a target immobilized on controlled pearl glass for easy protein recovery. A reference spectrum with pure, target-free resin must be recorded to rule out ligand–resin inter- actions. Relative binding strengths of competitive ligands can be assessed from a comparison of STD integrals. Structural information, however, is restricted to the binding epitope of the ligand. 38. Dalvit C, Pevarello P, Tato M, Veronesi M, Vulpetti A, Sundström M: Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J Biomol NMR 2000, 18:65-68. 39. 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Ligand binding is detected by signal broadening and extinction on the free form of a temporarily binding ligand. The strong magnetic marker allows a drastic reduction in the required protein and ligand concentrations, which also translates into lower minimum ligand solubilities and access to lower binding affinities. 42. Koenig BW, Mitchell DC, König S, Grzesiek S, Litman BJ, Bax A: Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin. J Biomol NMR 2000, 16:121-125. 43. Thompson GS, Shimizu H, Homans SW, Donohue-Rolfe A: Localization of the binding site for the oligosaccharide moiety of GB3 on verotoxin 1 using NMR residual dipolar coupling measurements. Biochemistry 2000, 39:13153-13156. 44. Hajduk PJ, Gerfin T, Boehlen J-M, Häberli M, Marek D, Fesik SW: • High-throughput nuclear magnetic resonance-based screening. J Med Chem 1999, 42:2315-2317. 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