Nmr in drug discovery

Key Points

  • NMR has become a valuable screening tool for analysing the binding of ligands to protein targets. Furthermore, NMR can provide structural information on protein–ligand interactions to aid in the optimization of weak-binding hits into high-affinity leads.

  • Methods for detecting binding fall into two main categories: those that monitor NMR signals from the ligand and those that monitor NMR signals from the protein.

  • Experiments that monitor the ligand exploit the large differences in the rates of rotational and translational motions of a small molecule in the free state relative to when it is bound to a macromolecules. The consequent effects on NMR properties, such as transverse and longitudinal relaxation times, are indicative of ligand binding.

  • Experiments that monitor the ligand have the advantages of requiring only small quantities of unlabelled protein, and also allowing several compounds to be studied simultaneously.

  • Experiments that monitor the protein, such as chemical-shift mapping, usually require labelled protein. However, coupled with resonance assignments, they can provide valuable information on the location of binding sites and the nature of the interactions that is not given by experiments that monitor the ligand.

  • In SAR by NMR, ligand binding is detected by chemical-shift mapping using a labelled protein target. In this way, small molecules that bind to two distinct sites on the protein are identified. Structural information on the binding modes and site positions is then used to aid the discovery of high-affinity compounds in which the two small-molecule fragments are linked.

  • SHAPES is a strategy in which ligand binding is assessed by observing signals from the ligand. Hits from a screen of a fairly small but diverse library of low-molecular weight scaffolds against an unlabelled protein target are optimized into high-affinity compounds by iterative synthetic modification and re-screening.

  • NMR-SOLVE exploits the fact that large families of proteins have adjacent binding sites, one of which is conserved throughout the family. It uses selective labelling of residues around the conserved binding site to guide the synthesis of high-affinity bi-ligand inhibitors, one part of which binds in the conserved binding site, and the other which binds in the adjacent site to give specificity.


NMR spectroscopy has evolved into an important technique in support of structure-based drug design. Here, we survey the principles that enable NMR to provide information on the nature of molecular interactions and, on this basis, we discuss current NMR-based strategies that can identify weak-binding compounds and aid their development into potent, drug-like inhibitors for use as lead compounds in drug discovery.

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Figure 1: Chemical-shift mapping.
Figure 2: NMR properties of macromolecules and small molecules.
Figure 3: SAR by NMR.
Figure 4: SHAPES screening.
Figure 5: NMR-SOLVE.


  1. 1

    Wüthrich, K. NMR of Proteins and Nucleic Acids. (Wiley, New York, 1986).

    Google Scholar 

  2. 2

    Pellecchia, M. et al. NMR-based structural characterization of large protein–ligand interactions. J. Biomol. NMR 22, 165–173 (2002).This paper describes the NMR-SOLVE method, which allows, with a suitable suite of NMR experiments and selective isotope-labelling schemes, the rapid structural characterization of ligand interactions with proteins of molecular masses up to 200 kDa.

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Medek, A., Hajduk, P. J., Mack, J. & Fesik, S. W. The use of differential chemical shift for determining the binding site location and orientation of protein-bound ligands. J. Am. Chem. Soc. 122, 1241–1242 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Abragam, A. Principles of Nuclear Magnetism (Oxford Univ. Press, New York, 1961).

    Google Scholar 

  5. 5

    Neuhaus, D. & Williamson, M. P. The Nuclear Overhauser Effect in Structural and Conformational Analysis. (Wiley–VCH, New York, 2000).

    Google Scholar 

  6. 6

    Kumar, A., Ernst, R. R. & Wüthrich, K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton–proton cross-relaxation networks in biological macromolecules. Biochem. Biophys. Res. Commun. 95, 1–6 (1980).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Hajduk, P. J., Olejniczak, E. T. & Fesik, S. W. One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules. J. Am. Chem. Soc. 119, 12257–12261 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Jahnke, W., Rüdisser, S. & Zurini, M. Spin label enhanced NMR screening. J. Am. Chem. Soc. 123, 3149–3150 (2001).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Ni, F. Recent developments in transferred NOE methods. Prog. NMR Spectrosc. 26, 517–606 (1994).

    CAS  Article  Google Scholar 

  10. 10

    Mayer, M. & Meyer, B. Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew. Chem. Int. Edn Engl. 38, 1784–1788 (1999).

    CAS  Article  Google Scholar 

  11. 11

    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. 121, 5336–5337 (1999).

    CAS  Article  Google Scholar 

  12. 12

    Chen, A. & Shapiro, M. J. NOE pumping: a novel NMR technique for identification of compounds with binding affinity to macromolecules. J. Am. Chem. Soc. 120, 10258–10259 (1998).

    CAS  Article  Google Scholar 

  13. 13

    Chen, A., & Shapiro, M. J. NOE pumping. 2. A high-throughput method to determine compounds with binding affinity to macromolecules by NMR. J. Am. Chem. Soc. 122, 414–415 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Dalvit, C. et al. Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J. Biomol. NMR 18, 65–68 (2000).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Gibbs, S. J. & Johnson, C. S. A PFG-NMR experiment for accurate diffusion and flow studies in the presence of Eddy currents. J. Magn. Reson. 93, 395–402 (1991).

    Google Scholar 

  16. 16

    Altieri, A. S., Hinton, D. P. & Byrd, R. A. Association of biomolecular systems via pulse-field gradient NMR self-diffusion measurements. J. Am. Chem. Soc. 117, 7566–7567 (1995).

    CAS  Article  Google Scholar 

  17. 17

    Shuker, S. B., Hajduk, P. J., Meadows, R. P. & Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534 (1996).A structure-based drug design strategy that uses NMR to design bi-ligand inhibitors that are specific for a given target. Relies initially on the complete structural characterization of protein–ligand complexes.

    CAS  Article  Google Scholar 

  18. 18

    Hajduk, P. J. et al. NMR-based discovery of lead inhibitors that block DNA-binding of the human papillomavirus protein E2 protein. J. Med. Chem. 40, 3144–3150 (1997).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Hajduk, P. J. et al. Discovery of potent non-peptide inhibitors of stromelysin using SAR by NMR. J. Am. Chem. Soc. 119, 5818–5827 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Hajduk, P. J. et al. Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis. J. Med. Chem. 42, 3852–3859 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Boehm, H. J. et al. Novel inhibitors of DNA girase: 3D structure based biased needle screening, hit validation by biophysical methods and 3D guided optimization. A promising alternative to random screening. J. Med. Chem. 43, 2664–2674 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Ross, A. & Senn, H. Automation of measurements and data evaluation in biomolecular NMR screening. Drug Discov. Today 6, 583–593 (2001).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Hajduk, P. J. et al. High-throughput nuclear magnetic resonance-based screening. J. Med. Chem. 42, 2315–2317 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Fejzo, J. et al. The SHAPES strategy: an NMR-based approach for lead generation in drug discovery. Chem. Biol. 6, 755–769 (1999).A drug design strategy that is based on the use of a library of low-molecular-mass fragments collected from the statistical analysis of established drugs. Protein targets are screened against weakly binding, drug-like molecular fragments, and hits are used to construct drug candidates computationally or in the laboratory.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Bemis, G. W. & Murcko, M. A. The properties of known drugs. 1. Molecular frameworks J. Med. Chem. 39, 2887–2893 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Hajduk, P. J. et al. NMR-based screening of proteins containing 13C-labeled methyl groups, J. Am. Chem. Soc. 122, 7898–7904 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Wüthrich, K. NMR in Structural Biology (World Scientific, Singapore, 1995).

    Google Scholar 

  28. 28

    Li, D. W., DeRose, E. F. & London, R. E. The inter-ligand Overhauser effect: a powerful new NMR approach for mapping structural relationships of macromolecular ligands. J. Biomol. NMR 15, 71–76 (1999).

    CAS  Article  PubMed  Google Scholar 

  29. 29

    London, R. E. Theoretical analysis of the inter-ligand Overhauser effect: a new approach for mapping structural relationships of macromolecular ligands. J. Magn. Reson. 141, 301–311 (1999).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Jahnke, W. et al. Second-site NMR screening with a spin-labeled first ligand. J. Am. Chem. Soc. 122, 7394–7395 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Meinecke, R. & Meyer, B. Determination of the binding specificity of an integral membrane protein by saturation transfer difference NMR: RGD peptide ligands binding to integrin IIb3. J. Med. Chem. 44, 3059–3065 (2001).

    CAS  Article  PubMed  Google Scholar 

  32. 32

    Fernández, C. et al. Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli. FEBS Lett. 504, 173–178 (2001).

    Article  PubMed  Google Scholar 

  33. 33

    Serber, Z., Ledwidge, R., Miller, S. M. & Dötsch, V. Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy. J. Am. Chem. Soc. 123, 8895–8901 (2001).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Bloch, F. Nuclear induction. Phys. Rev. 70, 460–474 (1946).

    CAS  Article  Google Scholar 

  35. 35

    Purcell, E. M., Torrey, E. C. & Pound, R. V. Resonance absorption by nuclear magnetic moments in a solid. Phys. Rev. 69, 37–38 (1946).

    CAS  Article  Google Scholar 

  36. 36

    The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

  37. 37

    Structural genomics. Nature Struct. Biol. 7 (Suppl.) (2000).

  38. 38

    Muchmore, D. C., McIntosh, L. P., Russel, C. B., Anderson, D. E. & Dahlquist, F. W. Expression and N-15 labeling of proteins for proton and 15N nuclear magnetic resonance. Methods Enzymol. 177, 44–73 (1989).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Kay, L. E. & Gardner, K. H. Solution NMR spectroscopy beyond 25 kDa. Curr. Opin. Struct. Biol. 7, 722–731 (1997).This paper describes advanced techniques for obtaining uniform and selective isotope labelling of proteins.

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Kay, L. E., Ikura, M., Tschudin, R. & Bax, A. Three-dimensional triple-resonance NMR-spectroscopy of isotopically enriched proteins. J. Magn. Reson. 89, 496–514 (1990).

    CAS  Google Scholar 

  41. 41

    Xu, R., Ayers, B., Cowburn, D. & Muir, T. W. Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies. Proc. Natl Acad. Sci. USA 96, 388–393 (1999).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    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 94, 12366–12371 (1997).Introduces the TROSY principle, which enables the use of solution NMR with high-molecular-weight proteins, and so expands the scope of NMR-based drug discovery approaches that rely on observation of a macromolecular target.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Wüthrich, K. The second decade — into the third millennium. Nature Struct. Biol. 5, 492–495 (1998).

    Article  PubMed  Google Scholar 

  44. 44

    Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Transverse relaxation-optimized spectroscopy (TROSY) for NMR studies of aromatic spin systems in 13C-labeled proteins. J. Am. Chem. Soc. 120, 6394–6400 (1998).

    CAS  Article  Google Scholar 

  45. 45

    Salzmann, M., Pervushin, K., Wider, G., Senn, H. & Wüthrich, K. NMR assignment and secondary structure determination of an octameric 110 kDa protein using TROSY in triple resonance experiments. J. Am. Chem. Soc. 122, 7543–7548 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Pellecchia, M., Sebbel, P., Hermanns, U., Glockshuber, R. & Wüthrich, K. Pilus chaperone FimC-adhesin FimH interactions mapped by TROSY-NMR. Nature Struct. Biol. 6, 336–339 (1999).

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Pellecchia, M. et al. SEA-TROSY (solvent exposed amides with TROSY): a method to resolve the problem of spectral overlap in very large proteins. J. Am. Chem. Soc. 123, 4633–4634 (2001).

    CAS  Article  PubMed  Google Scholar 

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cytochrome P450 reductase




NMR spectroscopy of proteins

 Protein Data Bank



The chemical shift of a particular nucleus is a measure of the dependence of the resonance frequency of the nucleus on its chemical environment, and is commonly indicated in parts per million (p.p.m.) relative to a reference compound.


An experiment that correlates different spins by scalar spin–spin coupling.


The process of attributing a resonance in an NMR spectrum to a particular nucleus in a molecule.


The application of radio-frequency irradiation alters the number of magnetic nuclei in each of their possible energy states in a magnetic field.


An NMR experiment that correlates three different spin types — typically 1H, 15N and 13C in biological macromolecules — through scalar spin–spin couplings to obtain resonance assignments.


A through-space interaction between different nuclear spins.


The chemical shift of a nuclear spin in a molecule varies with the orientation of the molecule relative to the external applied field.


Changes in the intensity of NMR signals, which are caused by through-space dipole–dipole coupling. Upper distance constraints obtained from 1H–1H NOEs are used for NMR structure determination of biological macromolecules.


Signal/noise ratios can be improved by reducing the operating temperature of some components of the NMR spectrometer.


A paramagnetic centre is characterized by the presence of localized unpaired electrons.

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Pellecchia, M., Sem, D. & Wüthrich, K. Nmr in drug discovery. Nat Rev Drug Discov 1, 211–219 (2002). https://doi.org/10.1038/nrd748

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