Fragment-based screening using X-ray crystallography and NMR spectroscopy

Current Opinion in Chemical Biology

Volumn 11, Issue 5, 2007, Pages 485-493

  Jhoti, Harren ^a   Cleasby, Anne ^a   Verdonk, Marcel ^a   Williams, Glyn ^a  

^a ASTEX PHARMACEUTICALS   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

AUTOMATION; COOLING; CRYSTALLIZATION; DATA ANALYSIS; DIFFRACTION; ENERGY TRANSFER; MOLECULAR INTERACTION; NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY; PROTEIN ANALYSIS; PROTEIN INTERACTION; PROTEIN STRUCTURE; PROTEIN TARGETING; REVIEW; STRUCTURE ANALYSIS; X RAY CRYSTALLOGRAPHY;

BINDING SITES; CRYSTALLOGRAPHY, X-RAY; DRUG EVALUATION, PRECLINICAL; LIGANDS; MAGNETIC RESONANCE SPECTROSCOPY; PROTEINS;

EID: 35348922285     PISSN: 13675931     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.cbpa.2007.07.010     Document Type: Review

References (66)

1. Congreve M., Murray C.W., and Blundell T.L. Structural biology and drug discovery. Drug Discov Today 10 (2005) 895-907
2. Rees D.C., Congreve M., Murray C.W., and Carr R. Fragment-based lead discovery. Nat Rev Drug Discov 3 (2004) 660-672
3. Erlanson D.A., McDowell R.S., and O'Brien T. Fragment-based drug discovery. J Med Chem 47 (2004) 3463-3482
4. Hajduk P.J., and Greer J. A decade of fragment-based drug design: strategic advances and lessons learned. Nat Rev Drug Discov 6 (2007) 211-219. This paper is a concise overview of the progress in, and current status of, fragment-based screening. It includes a list of published fragment-based inhibitors and a comparison of FBS and HTS for the generation of potent leads against 45 protein targets.
5. Hann M.M., Leach A.R., and Harper G. Molecular complexity and its impact on the probability of finding leads for drug discovery. J Chem Inf Comput Sci 41 (2001) 856-864
6. Hajduk P.J. Fragment-based drug design: how big is too big?. J Med Chem 49 (2006) 6972-6976. This paper reports a retrospective analysis of the potency increases that were observed during the elaboration of 18 optimised fragments into drug leads, against 15 diverse protein targets. The paper highlights the importance of starting with an optimised set of interactions and suggests that early data can be used successfully to predict the outcome of an optimisation.
7. Murray C.W., and Verdonk M.L. The consequences of translational and rotational entropy lost by small molecules on binding to proteins. J Comput Aided Mol Des 16 (2002) 741-753
8. Cele A.Z., and Metz J.T. Ligand efficiency indices as guideposts for drug discovery. Drug Discov Today 10 (2005) 464-469
9. Hopkins A.L., Groom C.R., and Alex A. Ligand efficiency: a useful metric for lead selection. Drug Discov Today 9 (2004) 430-431
10. Babaoglu K., and Shoichet B.K. Deconstructing fragment-based inhibitor discovery. Nat Chem Biol 2 (2006) 720-723
11. Hajduk P.J., Huth J.R., and Tse C. Predicting protein druggability. Drug Discov Today 10 (2005) 1675-1682
12. Ciulli A., Williams G., Smith A.G., Blundell T.L., and Abell C. Probing hot spots at protein-ligand binding sites: a fragment-based approach using biophysical methods. J Med Chem 49 (2006) 4992-5000
13. Keiser M.J., Roth B.L., Armbruster B.N., Ernsberger P., Irwin J.J., and Shoichet B.K. Relating protein pharmacology by ligand chemistry. Nat Biotechnol 25 (2007) 197-206
14. Mercier K.A., Baran M., Ramanathan V., Revesz P., Xiao R., Montelione G.T., and Powers R. FAST-NMR: functional annotation screening technology using NMR spectroscopy. J Am Chem Soc 128 (2006) 15292-15299
15. In: Jhoti H., and Leach A.R. (Eds). Structure-based Drug Discovery (2007), Springer
16. In: Jahnke W., and Erlanson D.A. (Eds). Fragment-based Approaches in Drug Discovery (2006), Wiley-VCH
17. Hartshorn M.J., Murray C.W., Cleasby A., Frederickson M., Tickle I.J., and Jhoti H. Fragment-based lead discovery using X-ray crystallography. J Med Chem 48 (2005) 403-413. This paper discusses the crystallographic approach to fragment-based screening used by Astex Therapeutics and presents results for several protein targets screened in this way. The method of soaking crystals in solutions containing cocktails of fragments is described.
18. Tickle I., Sharff A., Vinkovic M., Yon J., and Jhoti H. High-throughput protein crystallography and drug discovery. Chem Soc Rev 33 (2004) 558-565
19. Nienaber V.L., Richardson P.L., Klighofer V., Bouska J.J., Giranda V.L., and Greer J. Discovering novel ligands for macromolecules using X-ray crystallographic screening. Nat Biotechnol 18 (2000) 1105-1108
20. Card G.L., Blasdel L., England B.P., Zhang C., Suzuki Y., Gillette S., Fong D., Ibrahim P.N., Artis D.R., Bollag G., et al. A family of phosphodiesterase inhibitors discovered by cocrystallography and scaffold-based drug design. Nat Biotechnol 23 (2005) 201-207. This paper contains a recent description of the use of co-crystallisation in fragment-based screening of a phosphodiesterase protein target. Three hundred and nineteen compounds, pre-filtered by assay, were studied at using this method. Previously, compound soaking methods have been more widely described in the literature.
21. Forstner M., Leder L., and Mayr L.M. Optimization of protein expression systems for modern drug discovery. Expert Rev Proteomics 4 (2007) 67-78
22. Puri M., Robin G., Cowieson N., Forwood J.K., Listwan P., Hu S.H., Guncar G., Huber T., Kellie S., Hume D.A., et al. Focusing in on structural genomics: the University of Queensland structural biology pipeline. Biomol Eng 23 (2006) 281-289
23. Berry I.M., Dym O., Esnouf R.M., Harlos K., Meged R., Perrakis A., Sussman J.L., Walter T.S., Wilson J., and Messerschmidt A. SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new technologies and future aspects. Acta Crystallogr D Biol Crystallogr 62 (2006) 1137-1149. This paper reviews progress in high-throughput and small-scale crystallisation which has been made as part of the SPINE project. The methods used for quality control of protein samples are discussed. The paper illustrates how the technology has advanced in recent years and summarises suppliers of equipment, including robots.
24. Lau B.T., Baitz C.A., Dong X.P., and Hansen C.L. A complete microfluidic screening platform for rational protein crystallization. J Am Chem Soc 129 (2007) 454-455
25. Walter T.S., Diprose J.M., Mayo C.J., Siebold C., Pickford M.G., Carter L., Sutton G.C., Berrow N.S., Brown J., Berry I.M., et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr D Biol Crystallogr 61 (2005) 651-657
26. Arzt S., Beteva A., Cipriani F., Delageniere S., Felisaz F., Forstner G., Gordon E., Launer L., Lavault B., Leonard G., et al. Automation of macromolecular crystallography beamlines. Prog Biophys Mol Biol 89 (2005) 124-152
27. Skarzynski T., and Thorpe J. Industrial perspective on X-ray data collection and analysis. Acta Crystallogr D Biol Crystallogr 62 (2006) 102-107
28. Muchmore S.W., Olson J., Jones R., Pan J., Blum M., Greer J., Merrick S.M., Magdalinos P., and Nienaber V.L. Automated crystal mounting and data collection for protein crystallography. Structure 8 (2000) R243-R246
29. Beteva A., Cipriani F., Cusack S., Delageniere S., Gabadinho J., Gordon E.J., Guijarro M., Hall D.R., Larsen S., Launer L., et al. High-throughput sample handling and data collection at synchrotrons: embedding the ESRF into the high-throughput gene-to-structure pipeline. Acta Crystallogr D Biol Crystallogr 62 (2006) 1162-1169. This paper gives an example of the recent progress and improvements in automation of data collection and handling at a synchrotron. It describes the data-collection system implemented on beamlines at the European Synchrotron Radiation Facility, which was developed as part of the Structural Proteomics in Europe (SPINE) programme.
30. Cork C., O'Neill J., Taylor J., and Earnest T. Advanced beamline automation for biological crystallography experiments. Acta Crystallogr D Biol Crystallogr 62 (2006) 852-858
31. Oldfield T.J. X-LIGAND: an application for the automated addition of flexible ligands into electron density. Acta Crystallogr D Biol Crystallogr 57 (2001) 696-705
32. Zwart P.H., Langer G.G., and Lamzin V.S. Modelling bound ligands in protein crystal structures. Acta Crystallogr D Biol Crystallogr 60 (2004) 2230-2239
33. Evrard G.X., Langer G.G., Perrakis A., and Lamzin V.S. Assessment of automatic ligand building in ARP/wARP. Acta Crystallogr D Biol Crystallogr 63 (2007) 108-117. This study examines the performance of the automatic ligand-placement module in ARP/wARP against a large test set of 3884 structures from the PDB. The algorithm uses a two-step approach: it first locates the position where the ligand should be placed, and then constructs the ligand in that position. Locating the position of the ligand is most successful for larger ligands, whereas the construction of the ligands in the density works better for smaller compounds.
34. Mooij W.T., Hartshorn M.J., Tickle I.J., Sharff A.J., Verdonk M.L., and Jhoti H. Automated protein-ligand crystallography for structure-based drug design. Chem Med Chem 1 (2006) 827-838. In this study, the authors present a completely integrated approach to solving the structures of protein-ligand complexes that involves data processing, protein/ligand refinement, water placement and the fitting of the ligand into the electron density. For the ligand placement, the docking program GOLD is used with a scoring function that reflects the overlap between observed and calculated electron density. The ligand-fitting methodology is shown to perform well on a test set of 40 PDB structures.
35. Jones G., Willett P., Glen R.C., Leach A.R., and Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267 (1997) 727-748
36. Gronquist M., Meinwald J., Eisner T., and Schroeder F.C. Exploring uncharted terrain in nature's structure space using capillary NMR spectroscopy: 13 steroids from 50 fireflies. J Am Chem Soc 127 (2005) 10810-10811
37. Jahnke W., Rudisser S., and Zurini M. Spin label enhanced NMR screening. J Am Chem Soc 123 (2001) 3149-3150
38. McCoy M.A., Senior M.M., and Wyss D.F. Screening of protein kinases by ATP-STD NMR spectroscopy. J Am Chem Soc 127 (2005) 7978-7979
39. Huth J.R., Sun C., Sauer D.R., and Hajduk P.J. Utilization of NMR-derived fragment leads in drug design. Meth Enzymol 394 (2005) 549-571. In this paper, the authors summarise their experiences at Abbott Laboratories in developing leads from NMR screening hits. The discussion encompasses first-site and second-site screening, fragment linking, fragment elaboration and core replacement strategies in which elaborated leads are modified using fragment data.
40. Shoichet B.K. Screening in a spirit haunted world. Drug Discov Today 11 (2006) 607-615. This study is an informative discussion of the effects of compound aggregation, oxidation or other reactivity on high-throughput screening. Many of the conclusions can be applied equally to fragments. However the biophysical techniques employed by fragment screening usually ensures that such false positives and false negatives can be reliably detected.
41. Sem D.S. NMR-guided fragment assembly. In: Jahnke W., and Erlanson D.A. (Eds). Fragment-based Approaches in Drug Discovery. Methods and Principles in Medicinal Chemistry vol 34 (2006), Wiley-VCH 149-180
42. Lepre C.A., and Moore J.M. Fragment-based NMR screening in lead discovery. In: Jhoti H., and Leach A.R. (Eds). Structure-based Drug Discovery (2007), Springer 73-98
43. Schanda P., and Brutscher B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127 (2005) 8014-8015
44. Frydman L., Lupulescu A., and Scherf T. Principles and features of single-scan two-dimensional NMR spectroscopy. J Am Chem Soc 125 (2003) 9204-9217
45. Feliz M., Garcia J., Aragon E., and Pons M. Fast 2D NMR ligand screening using Hadamard spectroscopy. J Am Chem Soc 128 (2006) 7146-7147
46. Wyss D.F., Arasappan A., Senior M.M., Wang Y.S., Beyer B.M., Njoroge F.G., and McCoy M.A. Non-peptidic small-molecule inhibitors of the single-chain hepatitis C virus NS3 protease/NS4A cofactor complex discovered by structure-based NMR screening. J Med Chem 47 (2004) 2486-2498
47. Wang B., and Merz Jr. K.M. Validation of the binding site structure of the cellular retinol-binding protein (CRBP) by ligand NMR chemical shift perturbations. J Am Chem Soc 127 (2005) 5310-5311
48. 13C-labeled methyl groups. J Am Chem Soc 122 (2000) 7898-7904
49. Constantine K.L., Davis M.E., Metzler W.J., Mueller L., and Claus B.L. Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments. J Am Chem Soc 128 (2006) 7252-7263. In this study, the authors demonstrate that sufficient information concerning the binding mode of a fragment to a protein target can be generated from protein-ligand NOE data, without the need for residue-specific NMR assignments. In three cases involving two protein targets, LFA-1 and mFABP, residue and atom-type NMR assignments based on chemical shifts were sufficient to identify the known binding modes with good accuracy. This would allow NMR data to be used to select and model fragment hits at an early stage of a drug-design project, even when investigating a novel target.
50. Dalvit C., Fagerness P.E., Hadden D.T., Sarver R.W., and Stockman B.J. Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability. J Am Chem Soc 125 (2003) 7696-7703
51. Papeo G., Giordano P., Brasca M.G., Buzzo F., Caronni D., Ciprandi F., Mongelli N., Veronesi M., Vulpetti A., and Dalvit C. Polyfluorinated amino acids for sensitive (19)F NMR-based screening and kinetic measurements. J Am Chem Soc 129 (2007) 5665-5672
52. Dalvit C., Ardini E., Fogliatto G.P., Mongelli N., and Veronesi M. Reliable high-throughput functional screening with 3-FABS. Drug Discov Today 9 (2004) 595-602
53. 1H NMR probe which can reduce protein consumption and increase throughput while still allowing the fragments to be characterised during screening.
54. Mayer M., and Meyer B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc 123 (2001) 6108-6117
55. Hajduk P.J., Mack J.C., Olejniczak E.T., Park C., Dandliker P.J., and Beutel B.A. SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes. J Am Chem Soc 126 (2004) 2390-2398
56. Sanchez-Pedregal V.M., Reese M., Meiler J., Blommers M.J., Griesinger C., and Carlomagno T. The INPHARMA method: protein-mediated interligand NOEs for pharmacophore mapping. Angew Chem Int Ed Engl 44 (2005) 4172-4175. This study reports that the INPHARMA method (interligand NOE for pharmacophore mapping) permits the determination of the relative orientations of two competitive fragment hits in a protein-binding site. The method can be applied to large proteins that are not isotopically labelled though the analysis of the NOE data requires a structural model for the binding site. The method is illustrated using epithilone A and baccatin III binding to tubulin.
57. Vanwetswinkel S., Heetebrij R.J., van Duynhoven J., Hollander J.G., Filippov D.V., Hajduk P.J., and Siegal G. TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery. Chem Biol 12 (2005) 207-216
58. Hajduk P.J., Huth J.R., and Fesik S.K. Druggability indices for protein targets derived from NMR-based screening data. J Med Chem 48 (2005) 2518-2525. In this study, the authors survey hit rates from protein-detected NMR screens of 28 binding sites from a diverse set of 23 proteins. The measured hit rates are compared with calculated properties of the active sites and the properties are parameterised in order to predict the probability of developing a drug-like ligand for these binding sites. The authors include a wealth of stimulating data in the supplementary material.
59. Blaney J., Nienaber V., and Burley S.K. Fragment-based lead discovery and optimisation using X-ray crystallography, computational chemistry, and high-throughput organic synthesis. In: Jahnke W., and Erlanson D.A. (Eds). Fragment-based Approaches in Drug Discovery. Methods and Principles in Medicinal Chemistry vol 34 (2006), Wiley-VCH 215-248
60. Murray C.W., Callaghan O., Chessari G., Cleasby A., Congreve M., Frederickson M., Hartshorn M.J., McMenamin R., Patel S., and Wallis N. Application of fragment screening by X-ray crystallography to beta-secretase. J Med Chem 50 (2007) 1116-1123. The application of crystallographic fragment screening to a drug target, β-secretase, generally thought to be intractable, is described here. Several different inhibitor chemotypes were identified by this method, one of which contained a recognition motif not previously seen for aspartyl proteases.
61. Oltersdorf T., Elmore S.W., Shoemaker A.R., Armstrong R.C., Augeri D.J., Belli B.A., Bruncko M., Deckwerth T.L., Dinges J., Hajduk P.J., et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435 (2005) 677-681. A biologically active inhibitor of the interaction between Bcl-2 and the BH3 helix of pro-apoptotic proteins has been constructed by linking fragments derived from NMR screening. This is the first published application of fragment-based screening to the inhibition of a protein-protein interaction.
62. Howard N., Abell C., Blakemore W., Chessari G., Congreve M., Howard S., Jhoti H., Murray C.W., Seavers L.C., and van Montfort R.L. Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors. J Med Chem 49 (2006) 1346-1355
63. Bosch J., Robien M.A., Mehlin C., Boni E., Riechers A., Buckner F.S., Van Voorhis W.C., Myler P.J., Worthey E.A., DeTitta G., et al. Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem 49 (2006) 5939-5946. In this study, Bosch et al. describe time-course experiments in soaking fragments into crystals. It is shown that, in some cases, timescales as short as 10 s can be used. This was done in order to limit damage done to fragile crystals, caused by long immersions in solutions containing some fragment cocktails.
64. Kumar A., Mandiyan V., Suzuki Y., Zhang C., Rice J., Tsai J., Artis D.R., Ibrahim P., and Bremer R. Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma. J Mol Biol 348 (2005) 183-193
65. Tsao D.H., Sutherland A.G., Jennings L.D., Li Y., Rush III T.S., Alvarez J.C., Ding W., Dushin E.G., Dushin R.G., Haney S.A., et al. Discovery of novel inhibitors of the ZipA/FtsZ complex by NMR fragment screening coupled with structure-based design. Bioorg Med Chem 14 (2006) 7953-7961
66. Bretonnet A.S., Jochum A., Walker O., Krimm I., Goekjian P., Marcillat O., and Lancelin J.M. NMR screening applied to the fragment-based generation of inhibitors of creatine kinase exploiting a new interaction proximate to the ATP binding site. J Med Chem 50 (2007) 1865-1875. This paper presents both water-LOGSY and STD-NMR data for selected fragments which bind to creatine kinase. The hits were identified from a library of only 53 fragments and efficiently elaborated into compounds with micromolar affinity, guided by docking studies only.