Publikationsserver der Universitätsbibliothek Marburg

Titel:Fragment based Drug Discovery; Design and Validation of a Fragment Library; Computer-based Fragment Screening and Fragment-to-Lead Expansion
Autor:Craan, Tobias Friedrich
Weitere Beteiligte: Klebe, Gerhard (Prof. Dr.)
URN: urn:nbn:de:hebis:04-z2011-04293
DDC: Naturwissenschaften
Titel (trans.):Fragment-basierte Wirkstoffentwicklung; Entwicklung und Validierung einer Fragmentbibliotek; Computer-basierte Fragment-Suchen und Fragment zu Leitstruktur Entwicklung


Fragment, Fragmentbibliotek, Fragment library, Fragmentbasierte Leitstrukturentwicklung, FBLD

In recent years, fragment screening has become a popular approach to identify new lead structures. Fragments are usually defined by the Astex ‘rule of three’ (RO3). Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance spectroscopy (NMR), biochemical assays and X-ray crystallography are efficient screening techniques to discover prospective fragments as binders. However, these methods need an assembled fragment library. We designed an in-house fragment library, starting from approx. 380,000 commercially available fragments. During library design, we modified the RO3 and we did no strict filtering of physico-chemical properties during fragment enumeration (e.g. twice the number of H-bond acceptors was allowed). The fragments were stepwise reduced to 4,000 compounds. The last step was a visual inspection of the candidates, which lead to a final fragment library of 364 fragments. To validate the quality of the library, we screened it against endothiapepsin. The biochemical screening suggested 55 hits, which were entered into a crystallographic screen. Eleven complex crystal structures were determined, pointing out the remarkably high hit rate of the designed library. HotspotsX is a program which predicts (based on knowledge-based potentials) the probability of a certain atom type at a certain position in the binding pocket of a target enzyme. The eleven crystal structures obtained before were used to validate the program HotspotsX. Due to chemical diversity and the different binding modes of the fragments observed for the library examples we obtained binding through aromatic- , H-bond donor- , acceptor- , doneptor- and hydrophobic interactions. The calculated HotspotsX maps coincide remarkably well with the crystallographically determined fragment positions inside the binding pocket. The program HotspotsX has also been validated with crystal structures of molecular probes like phenol, urea and methylurea. Crystal structures of these molecular probes were determined with different targets. Overall, the experimental hotspot analysis coincided well with the computed contour maps. Thus, the calculated maps by HotspotsX have an excellent predictive power. Based on the binding modes of the molecular probe phenol to the cAMP-dependent protein kinase A (PKA), we started a fragment growing approach. In the latter complex, three phenol molecules are bound. Two are occupying the ATP binding site and one is sitting on top of the glycine-rich loop (G-loop). A virtual screening, using the hinge binding phenol as constraint, suggested a phenol derivative for which a crystal structure could be determined. Starting from this hit, a hotspot analysis was performed. This analysis indicates that growth in the direction of the G-loop, placing an aromatic portion under the G-loop and an acceptor functionality capable to address Lys72 is desired. The first compound of this de novo design had an affinity of 70 µM. In the following first design cycle, we were able to enhance the affinity to 6.5 µM. In the second design cycle an additional amino function was introduced, which did not improve affinity dramatically, but enhanced ligand efficiency to 0.38. In the last cycle, a spacer of one and two methylene groups was introduced and the affinity could be increased to about 110 nM for a diastereomeric mixture of four compounds. The phenol-PKA complex provides a putative allosteric site of PKA. The G-loop in this structure is in a closed state which is stabilized by two H-bonds. This G-loop conformation is probably induced by the phenol molecule sitting on top of the G-loop. Therefore, several molecular dynamics (MD) studies were performed, lacking different phenol molecules, to get insights into the G-loop opening. The MD studies suggest that after removal of the phenol sitting on top of the G-loop some first side chain movements are initiated that can indicate the first steps of the G-loop opening cascade. In a different project, a virtual screening approach was used to find new inhibitors for aldose reductase. A pre-filtered subset of the ZINC database was used as ligand dataset. For the best hit, a series of five compounds was synthesized. Among them one compound displayed an inhibition of 920 nM. The available assays to detect fragment hits are currently not sufficient. The challenges are the low affinity of the fragments and their poor solubility. Therefore, the known thermal shift assay was applied and adapted to detect fragment hits. To validate the method, it was used to characterize variant mutations of EctD. Lastly, a modeling study was used to get ideas about possible binding modes of arachidonic acid derivatives in a K+ ion channel. One predominant binding pose could not be suggested. The study proposes, however, that one arachidonic acid molecule can occupy the inner pore cavity, which is consistent with experimental data.

Bibliographie / References

  1. AmberTools. 129. Manchester. Contributed parameters 130. Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. Antechamber, An Accessory Software Package For Molecular Mechanical Calculations. 131. GAFF. 132. Gnuplot.
  2. McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J. Likelihood- enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr 2005, 61, 458- 64.
  3. Howard, S.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D. M.; Curry, J.; Devine, L. A.; Early, T. R.; Fazal, L.; Gill, A. L.; Heathcote, M.; Maman, S.; Matthews, J. E.; McMenamin, R. L.; Navarro, E. F.; O'Brien, M. A.; O'Reilly, M.; Rees, D. C.; Reule, M.; Tisi, D.; Williams, G.; Vinkovic, M.; Wyatt, P. G. Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor with potent aurora kinase activity. J Med Chem 2009, 52, 379-88.
  4. X-ray, neutron and NMR studies of the catalytic mechanism of aspartic proteinases.
  5. de Kloe, G. E.; Bailey, D.; Leurs, R.; de Esch, I. J. Transforming fragments into candidates: small becomes big in medicinal chemistry. Drug Discov Today 2009, 14, 630-46.
  6. Clifton, I. J.; McDonough, M. A.; Ehrismann, D.; Kershaw, N. J.; Granatino, N.; Schofield, C. J. Structural studies on 2-oxoglutarate oxygenases and related double- stranded beta-helix fold proteins. J Inorg Biochem 2006, 100, 644-69.
  7. Abad-Zapatero, C.; Metz, J. T. Ligand efficiency indices as guideposts for drug discovery. Drug Discov Today 2005, 10, 464-9.
  8. Makara, G. M. On sampling of fragment space. J Med Chem 2007, 50, 3214-21.
  9. Roach, P. L.; Clifton, I. J.; Fulop, V.; Harlos, K.; Barton, G. J.; Hajdu, J.; Andersson, I.; Schofield, C. J.; Baldwin, J. E. Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes. Nature 1995, 375, 700-4.
  10. English, A. C.; Groom, C. R.; Hubbard, R. E. Experimental and computational mapping of the binding surface of a crystalline protein. Protein Engineering. 2001, 14, 47-59.
  11. Madhusudan; Akamine, P.; Xuong, N. H.; Taylor, S. S. Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase. Nat Struct Biol 2002, 9, 273-7.
  12. Kramer, C.; Heinisch, T.; Fligge, T.; Beck, B.; Clark, T. A consistent dataset of kinetic solubilities for early-phase drug discovery. ChemMedChem 2009, 4, 1529-36.
  13. Kramer, C.; Beck, B.; Clark, T. Insolubility classification with accurate prediction probabilities using a MetaClassifier. J Chem Inf Model 50, 404-14.
  14. Baurin, N.; Aboul-Ela, F.; Barril, X.; Davis, B.; Drysdale, M.; Dymock, B.; Finch, H.; Fromont, C.; Richardson, C.; Simmonite, H.; Hubbard, R. E. Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets. J Chem Inf Comput Sci 2004, 44, 2157-66.
  15. Elkins, J. M.; Hewitson, K. S.; McNeill, L. A.; Seibel, J. F.; Schlemminger, I.; Pugh, C. W.; Ratcliffe, P. J.; Schofield, C. J. Structure of factor-inhibiting hypoxia- inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1 alpha. J Biol Chem 2003, 278, 1802-6.
  16. Decher, N.; Gonzalez, T.; Streit, A. K.; Sachse, F. B.; Renigunta, V.; Soom, M.; Heinemann, S. H.; Daut, J.; Sanguinetti, M. C. Structural determinants of Kvbeta1.3- induced channel inactivation: a hairpin modulated by PIP2. EMBO J 2008, 27, 3164-74.
  17. Goodford, P. J. A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J Med Chem 1985, 28, 849-57.
  18. Medina, J. s. R.; Blackledge, C. W.; Heerding, D. A.; Campobasso, N.; Ward, P.; Briand, J.; Wright, L.; Axten, J. M. Aminoindazole PDK1 Inhibitors: A Case Study in Fragment-Based Drug Discovery. ACS Medicinal Chemistry Letters 1, 439-442. 35. Nordstrom, H.; Gossas, T.; Hamalainen, M.; Kallblad, P.; Nystrom, S.; Wallberg, H.; Danielson, U. H. Identification of MMP-12 inhibitors by using biosensor-based screening of a fragment library. J Med Chem 2008, 51, 3449-59.
  19. Jhoti, H. A new school for screening. Nat Biotechnol 2005, 23, 184-6.
  20. Allen, K. N.; Bellamacina, C. R.; Ding, X.; Jeffrey, C. J.; Mattos, C. An Experimental Approach to Mapping the Binding Surfaces of Crystalline Proteins. The Journal of Physical Chemistry 1996, 100, 2605 -2611.
  21. Edwards, P. D.; Albert, J. S.; Sylvester, M.; Aharony, D.; Andisik, D.; Callaghan, O.; Campbell, J. B.; Carr, R. A.; Chessari, G.; Congreve, M.; Frederickson, M.; Folmer, R. H.; Geschwindner, S.; Koether, G.; Kolmodin, K.; Krumrine, J.; Mauger, R. C.; Murray, C. W.; Olsson, L. L.; Patel, S.; Spear, N.; Tian, G. Application of fragment- based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency. J Med Chem 2007, 50, 5912-25.
  22. Application of fragment-based NMR screening, X-ray crystallography, structure-based Literature 171 design, and focused chemical library design to identify novel microM leads for the development of nM BACE-1 (beta-site APP cleaving enzyme 1) inhibitors. J Med Chem 53, 942-50.
  23. Congreve, M.; Aharony, D.; Albert, J.; Callaghan, O.; Campbell, J.; Carr, R. A.; Chessari, G.; Cowan, S.; Edwards, P. D.; Frederickson, M.; McMenamin, R.; Murray, C. W.; Patel, S.; Wallis, N. Application of fragment screening by X-ray crystallography to the discovery of aminopyridines as inhibitors of beta-secretase. J Med Chem 2007, 50, 1124-32.
  24. Congreve, M.; Carr, R.; Murray, C.; Jhoti, H. A 'rule of three' for fragment-based lead discovery? Drug Discov Today 2003, 8, 876-7.
  25. Owens, J. Chris Lipinski discusses life and chemistry after the Rule of Five. Drug Discovery Today 2003, 8, 12-16.
  26. Pavel, E. G.; Zhou, J.; Busby, R. W.; Gunsior, M.; Townsend, C. A.; Solomon, E. I. Circular Dichroism and Magnetic Circular Dichroism Spectroscopic Studies of the Non-Heme Ferrous Active Site in Clavaminate Synthase and Its Interaction with α- Ketoglutarate Cosubstrate. Journal of the American Chemical Society 1998, 120, 743- 753.
  27. Blasiak, L. C.; Vaillancourt, F. H.; Walsh, C. T.; Drennan, C. L. Crystal structure of the non-haem iron halogenase SyrB2 in syringomycin biosynthesis. Nature 2006, 440, 368-71. 158. Hewitson, K. S.; Holmes, S. L.; Ehrismann, D.; Hardy, A. P.; Chowdhury, R.; Schofield, C. J.; McDonough, M. A. Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH). J Biol Chem 2008, 283, 25971-8.
  28. Yu, B.; Edstrom, W. C.; Benach, J.; Hamuro, Y.; Weber, P. C.; Gibney, B. R.; Hunt, J. F. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB. Nature 2006, 439, 879-84.
  29. Lubbers, T.; Angehrn, P.; Gmunder, H.; Herzig, S. Design, synthesis, and structure-activity relationship studies of new phenolic DNA gyrase inhibitors. Bioorg Med Chem Lett 2007, 17, 4708-14.
  30. Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Discovering high- affinity ligands for proteins: SAR by NMR. Science 1996, 274, 1531-4.
  31. Huth, J. R.; Park, C.; Petros, A. M.; Kunzer, A. R.; Wendt, M. D.; Wang, X.; Lynch, C. L.; Mack, J. C.; Swift, K. M.; Judge, R. A.; Chen, J.; Richardson, P. L.; Jin, S.; Tahir, S. K.; Matayoshi, E. D.; Dorwin, S. A.; Ladror, U. S.; Severin, J. M.; Walter, K. A.; Bartley, D. M.; Fesik, S. W.; Elmore, S. W.; Hajduk, P. J. Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies. Chem Biol Drug Des 2007, 70, 1-12.
  32. Liu, G.; Szczepankiewicz, B. G.; Pei, Z.; Janowick, D. A.; Xin, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Liang, H.; Hutchins, C. W.; Fesik, S. W.; Ballaron, S. J.; Stashko, M. A.; Lubben, T.; Mika, A. K.; Zinker, B. A.; Trevillyan, J. M.; Jirousek, M. R. Discovery and structure-activity relationship of oxalylarylaminobenzoic acids as inhibitors of protein tyrosine phosphatase 1B. J Med Chem 2003, 46, 2093-103.
  33. E.; Cosme, J.; Graham, B.; Day, P. J.; Downham, R.; Fazal, L.; Feltell, R.; Figueroa, E.; Frederickson, M.; Lewis, J.; McMenamin, R.; Murray, C. W.; O'Brien, M. A.; Parra, L.; Patel, S.; Phillips, T.; Rees, D. C.; Rich, S.; Smith, D. M.; Trewartha, G.; Vinkovic, M.; Williams, B.; Woolford, A. J. Discovery of (2,4-dihydroxy-5-isopropylphenyl)-[5-(4- methylpiperazin-1-ylmethyl)-1,3-di hydroisoindol-2-yl]methanone (AT13387), a novel inhibitor of the molecular chaperone Hsp90 by fragment based drug design. J Med Chem 53, 5956-69. 31. Caldwell, J. J.; Davies, T. G.; Donald, A.; McHardy, T.; Rowlands, M. G.; Aherne, G. W.; Hunter, L. K.; Taylor, K.; Ruddle, R.; Raynaud, F. I.; Verdonk, M.; Workman, P.; Garrett, M. D.; Collins, I. Identification of 4-(4-aminopiperidin-1-yl)-7H- pyrrolo[2,3-d]pyrimidines as selective inhibitors of protein kinase B through fragment elaboration. J Med Chem 2008, 51, 2147-57.
  34. J. Discovery of a novel Hsp90 inhibitor by fragment linking. ChemMedChem 5, 1697- 700.
  35. Geschwindner, S.; Olsson, L. L.; Albert, J. S.; Deinum, J.; Edwards, P. D.; de Beer, T.; Folmer, R. H. Discovery of a novel warhead against beta-secretase through fragment-based lead generation. J Med Chem 2007, 50, 5903-11.
  36. K.; Nilakantan, R.; Mosyak, L. Discovery of novel inhibitors of the ZipA/FtsZ complex by NMR fragment screening coupled with structure-based design. Bioorg Med Chem 2006, 14, 7953-61.
  37. Velec, H. F. G.; Gohlke, H.; Klebe, G. DrugScoreCSDKnowledge-Based Scoring Function Derived from Small Molecule Crystal Data with Superior Recognition Rate of Near-Native Ligand Poses and Better Affinity Prediction. Journal of Medicinal Chemistry 2005, 48, 6296-6303.
  38. Akamine, P.; Madhusudan; Wu, J.; Xuong, N. H.; Ten Eyck, L. F.; Taylor, S. S. Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure. J Mol Biol 2003, 327, 159-71.
  39. Majeux, N.; Scarsi, M.; Caflisch, A. Efficient electrostatic solvation model for protein-fragment docking. Proteins 2001, 42, 256-68. 98. Goodford, P. J. A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. Journal of Medicinal Chemistry 1985, 28, 849-857.
  40. Majeux, N.; Scarsi, M.; Apostolakis, J.; Ehrhardt, C.; Caflisch, A. Exhaustive docking of molecular fragments with electrostatic solvation. Proteins 1999, 37, 88-105.
  41. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001, 46, 3-26.
  42. Neudert, G.; Klebe, G. fconv: format conversion, manipulation, and feature computation of molecular data. Bioinformatics.
  43. Hausinger, R. P. FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit Rev Biochem Mol Biol 2004, 39, 21-68.
  44. Coates, L.; Erskine, P. T.; Crump, M. P.; Wood, S. P.; Cooper, J. B. Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism. J Mol Biol 2002, 318, 1405-15.
  45. Jahnke, W. Fragment-based approaches in drug discovery. Wiley-VCH-Verl.: Weinheim, 2006; p XXII, 369 S.
  46. Erlanson, D. A.; McDowell, R. S.; O'Brien, T. Fragment-based drug discovery. J Med Chem 2004, 47, 3463-82.
  47. Rees, D. C.; Congreve, M.; Murray, C. W.; Carr, R. Fragment-based lead discovery. Nat Rev Drug Discov 2004, 3, 660-72.
  48. Erlanson, D. A. Fragment-based lead discovery: a chemical update. Curr Opin Biotechnol 2006, 17, 643-52.
  49. Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Frederickson, M.; Tickle, I. J.; Jhoti, H. Fragment-based lead discovery using X-ray crystallography. J Med Chem 2005, 48, 403-13.
  50. Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Hutchins, C. W.; Zhao, H.; Lubben, T. H.; Ballaron, S. J.; Haasch, D. L.; Kaszubska, W.; Rondinone, C. M.; Trevillyan, J. M.; Jirousek, M. R. Fragment screening and assembly: a highly efficient approach to a selective and cell active protein tyrosine phosphatase 1B inhibitor. J Med Chem 2003, 46, 4232-5.
  51. Leach, A. R.; Hann, M. M.; Burrows, J. N.; Griffen, E. J. Fragment screening: an introduction. Mol Biosyst 2006, 2, 430-46.
  52. Zartler, E. R.; Shapiro, M. J. Fragonomics: fragment-based drug discovery. Curr Opin Chem Biol 2005, 9, 366-70.
  53. Miranker, A.; Karplus, M. Functionality maps of binding sites: a multiple copy simultaneous search method. Proteins 1991, 11, 29-34.
  54. Blundell, T. L.; Jhoti, H.; Abell, C. High-throughput crystallography for lead discovery in drug design. Nat Rev Drug Discov 2002, 1, 45-54.
  55. Tickle, I.; Sharff, A.; Vinkovic, M.; Yon, J.; Jhoti, H. High-throughput protein crystallography and drug discovery. Chem Soc Rev 2004, 33, 558-65.
  56. Blundell, T. L.; Patel, S. High-throughput X-ray crystallography for drug discovery. Curr Opin Pharmacol 2004, 4, 490-6.
  57. Keseru, G. M.; Makara, G. M. Hit discovery and hit-to-lead approaches. Drug Discov Today 2006, 11, 741-8.
  58. Saxty, G.; Woodhead, S. J.; Berdini, V.; Davies, T. G.; Verdonk, M. L.; Wyatt, P. G.; Boyle, R. G.; Barford, D.; Downham, R.; Garrett, M. D.; Carr, R. A. Identification of inhibitors of protein kinase B using fragment-based lead discovery. J Med Chem 2007, 50, 2293-6.
  59. Welford, R. W.; Kirkpatrick, J. M.; McNeill, L. A.; Puri, M.; Oldham, N. J.; Schofield, C. J. Incorporation of oxygen into the succinate co-product of iron(II) and 2- oxoglutarate dependent oxygenases from bacteria, plants and humans. FEBS Lett 2005, 579, 5170-4.
  60. Casale, E.; Collyer, C.; Ascenzi, P.; Balliano, G.; Milla, P.; Viola, F.; Fasano, M.; Menegatti, E.; Bolognesi, M. Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by 4',6-diamidino-2-phenylindole, 6- amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study. Biophys Chem 1995, 54, 75-81.
  61. Oblak, M.; Grdadolnik, S. G.; Kotnik, M.; Jerala, R.; Filipic, M.; Solmajer, T. In silico fragment-based discovery of indolin-2-one analogues as potent DNA gyrase inhibitors. Bioorg Med Chem Lett 2005, 15, 5207-10.
  62. Siegal, G.; Ab, E.; Schultz, J. Integration of fragment screening and library design. Drug Discov Today 2007, 12, 1032-9.
  63. Branden, C.; Tooze, J. Introduction to protein structure. 2. ed.; Garland: New York , NY, 1999; p XIV, 410 S.
  64. Oprea, T. I.; Davis, A. M.; Teague, S. J.; Leeson, P. D. Is There a Difference between Leads and Drugs? A Historical Perspective. Journal of Chemical Information and Computer Sciences 2001, 41, 1308-1315.
  65. Gohlke, H.; Hendlich, M.; Klebe, G. Knowledge-based scoring function to predict protein-ligand interactions. Journal of Molecular Biology 2000, 295, 337-356. 101. Neudert, G.; Klebe, G. fconv: format conversion, manipulation and feature computation of molecular data. Bioinformatics 27, 1021-2.
  66. Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430-431.
  67. English, A. C.; Done, S. H.; Caves, L. S.; Groom, C. R.; Hubbard, R. E. Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol. Proteins 1999, 37, 628-640.
  68. F.; Sowadski, J. M. 2.2 A refined crystal structure of the catalytic subunit of cAMP- dependent protein kinase complexed with MnATP and a peptide inhibitor. Acta Crystallographica Section D 1993, 49, 362-365.
  69. Nettleship, J. E.; Brown, J.; Groves, M. R.; Geerlof, A. Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering. Methods Mol Biol 2008, 426, 299-318.
  70. Carrell, H. L.; Hoier, H.; Glusker, J. P. Modes of binding substrates and their analogues to the enzyme d-xylose isomerase. Acta Crystallographica Section D 1994, 50, 113-123.
  71. Hann, M. M.; Leach, A. R.; Harper, G. Molecular complexity and its impact on the probability of finding leads for drug discovery. J Chem Inf Comput Sci 2001, 41, 856-64.
  72. Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. J Mol Biol 1995, 245, 43-53.
  73. Mattos, C.; Bellamacina, C. R.; Peisach, E.; Pereira, A.; Vitkup, D.; Petsko, G. A.; Ringe, D. Multiple solvent crystal structures: probing binding sites, plasticity and hydration. J Mol Biol. 2006, 357, 1471-1482.
  74. Gunera, J. New Scaffolds for Aldo-Keto Reductase: A Virtual Screening Study. Bachelor, Philipps Universität Marburg, Marburg, 2010
  75. Dalvit, C.; Fasolini, M.; Flocco, M.; Knapp, S.; Pevarello, P.; Veronesi, M. NMR- Based screening with competition water-ligand observed via gradient spectroscopy experiments: detection of high-affinity ligands. J Med Chem 2002, 45, 2610-4.
  76. Bursy, J.; Pierik, A. J.; Pica, N.; Bremer, E. Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. J Biol Chem 2007, 282, 31147-55.
  77. Larson, M. K.; Whitaker, J. R. Endothia parasitica Protease. Parameters Affecting Stability of the Rennin-like Enzyme. Journal of Dairy Science 1970, 53, 262-269.
  78. Matter, H.; Nazare, M.; Gussregen, S.; Will, D. W.; Schreuder, H.; Bauer, A.; Urmann, M.; Ritter, K.; Wagner, M.; Wehner, V. Evidence for C-Cl/C-Br...pi interactions as an important contribution to protein-ligand binding affinity. Angew Chem Int Ed Engl 2009, 48, 2911-6.
  79. Otwinowski, Z., & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology 1997, 276, 307-326.
  80. Donald, A.; McHardy, T.; Rowlands, M. G.; Hunter, L. J.; Davies, T. G.; Berdini, V.; Boyle, R. G.; Aherne, G. W.; Garrett, M. D.; Collins, I. Rapid evolution of 6- phenylpurine inhibitors of protein kinase B through structure-based design. J Med Chem 2007, 50, 2289-92. 33. Frederickson, M.; Callaghan, O.; Chessari, G.; Congreve, M.; Cowan, S. R.; Matthews, J. E.; McMenamin, R.; Smith, D. M.; Vinkovic, M.; Wallis, N. G. Fragment- based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase- type plasminogen activator. J Med Chem 2008, 51, 183-6.
  81. Brown, D.; Superti-Furga, G. Rediscovering the sweet spot in drug discovery. Drug Discovery Today 2003, 8, 1067-1077.
  82. Swayze, E. E.; Jefferson, E. A.; Sannes-Lowery, K. A.; Blyn, L. B.; Risen, L. M.; Arakawa, S.; Osgood, S. A.; Hofstadler, S. A.; Griffey, R. H. SAR by MS: a ligand based technique for drug lead discovery against structured RNA targets. J Med Chem 2002, 45, 3816-9.
  83. Selective protein tyrosine phosphatase 1B inhibitors: targeting the second phosphotyrosine binding site with non-carboxylic acid-containing ligands. J Med Chem 2003, 46, 3437-40.
  84. Sheldrick, G. M.; Schneider, T. R. SHELXL: high-resolution refinement. Methods Enzymol 1997, 277, 319-43.
  85. Holland, D. R.; Tronrud, D. E.; Pley, H. W.; Flaherty, K. M.; Stark, W.; Jansonius, J. N.; McKay, D. B.; Matthews, B. W. Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis. Biochemistry 1992, 31, 11310-11316.
  86. Sutherland, A. G.; Alvarez, J.; Ding, W.; Foreman, K. W.; Kenny, C. H.; Labthavikul, P.; Mosyak, L.; Petersen, P. J.; Rush, T. S., 3rd; Ruzin, A.; Tsao, D. H.; Wheless, K. L. Structure-based design of carboxybiphenylindole inhibitors of the ZipA- FtsZ interaction. Org Biomol Chem 2003, 1, 4138-40.
  87. M.; Cane, D. E.; Noel, J. P. Structure of 4-diphosphocytidyl-2-C-methylerythritol synthetase involved in mevalonate-independent isoprenoid biosynthesis. Nature Structural Molecular Biolology 2001, 8, 641-648.
  88. Bruncko, M.; Oost, T. K.; Belli, B. A.; Ding, H.; Joseph, M. K.; Kunzer, A.; Martineau, D.; McClellan, W. J.; Mitten, M.; Ng, S. C.; Nimmer, P. M.; Oltersdorf, T.; Park, C. M.; Petros, A. M.; Shoemaker, A. R.; Song, X.; Wang, X.; Wendt, M. D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL. J Med Chem 2007, 50, 641-62.
  89. Verdonk, M. L.; Cole, J. C.; Taylor, R. SuperStar: a knowledge-based approach for identifying interaction sites in proteins. J Mol Biol 1999, 289, 1093-108.
  90. Koski, M. K.; Hieta, R.; Bollner, C.; Kivirikko, K. I.; Myllyharju, J.; Wierenga, R. K. The active site of an algal prolyl 4-hydroxylase has a large structural plasticity. J Biol Chem 2007, 282, 37112-23.
  91. Allen, F. H. The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr B 2002, 58, 380-8.
  92. Purpero, V.; Moran, G. R. The diverse and pervasive chemistries of the alpha-keto acid dependent enzymes. J Biol Inorg Chem 2007, 12, 587-601.
  93. Flashman, E.; Schofield, C. J. The most versatile of all reactive intermediates? Nat Chem Biol 2007, 3, 86-7.
  94. Lepre, C. A.; Moore, J. M.; Peng, J. W. Theory and Applications of NMR-Based Screening in Pharmaceutical Research. Chemical Reviews 2004, 104, 3641-3676.
  95. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 2006, 357, 289-98.
  96. Fejzo, J.; Lepre, C. A.; Peng, J. W.; Bemis, G. W.; Ajay; Murcko, M. A.; Moore, J. M. The SHAPES strategy: an NMR-based approach for lead generation in drug discovery. Chem Biol 1999, 6, 755-69.
  97. Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2007, 2, 2212-21.
  98. Joseph-McCarthy, D.; Hogle, J. M.; Karplus, M. Use of the multiple copy simultaneous search (MCSS) method to design a new class of picornavirus capsid binding drugs. Proteins 1997, 29, 32-58.
  99. Ringe, D. What makes a binding site a binding site? Curr Opin Struct Biol. 1995, 5, 825-829.
  100. Klebe, G. Wirkstoffdesign : Entwurf und Wirkung von Arzneistoffen. 2. Aufl.. ed.; Spektrum Akad. Verl.: Heidelberg, 2009; p XX, 634 S.
  101. Cooper, J.; Quail, W.; Frazao, C.; Foundling, S. I.; Blundell, T. L.; Humblet, C.; Lunney, E. A.; Lowther, W. T.; Dunn, B. M. X-ray crystallographic analysis of inhibition of endothiapepsin by cyclohexyl renin inhibitors. Biochemistry 1992, 31, 8142-50.
  102. Dymock, B. W.; Barril, X.; Brough, P. A.; Cansfield, J. E.; Massey, A.; McDonald, E.; Hubbard, R. E.; Surgenor, A.; Roughley, S. D.; Webb, P.; Workman, P.; Wright, L.; Drysdale, M. J. Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design. J Med Chem 2005, 48, 4212-5.
  103. Cheung, K. M.; Matthews, T. P.; James, K.; Rowlands, M. G.; Boxall, K. J.; Sharp, S. Y.; Maloney, A.; Roe, S. M.; Prodromou, C.; Pearl, L. H.; Aherne, G. W.; McDonald, E.; Workman, P. The identification, synthesis, protein crystal structure and in vitro biochemical evaluation of a new 3,4-diarylpyrazole class of Hsp90 inhibitors. Bioorg Med Chem Lett 2005, 15, 3338-43.
  104. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. A fast flexible docking method using an incremental construction algorithm. J Mol Biol 1996, 261, 470-89. 122. Gerlach, C.; Munzel, M.; Baum, B.; Gerber, H. D.; Craan, T.; Diederich, W. E.; Klebe, G. KNOBLE: a knowledge-based approach for the design and synthesis of readily accessible small-molecule chemical probes to test protein binding. Angew Chem Int Ed Engl 2007, 46, 9105-9. 123. 124. Huggenvik, J. I.; Collard, M. W.; Stofko, R. E.; Seasholtz, A. F.; Uhler, M. D. Regulation of the Human Enkephalin Promoter by Two Isoforms of the Catalytic Subunit of Cyclic Adenosine 3',5'-Monophosphate-Dependent Protein Kinase. Mol Endocrinol 1991, 5, 921-930. 125. Gjertsen, B. T.; Døskeland, S. O. Protein phosphorylation in apoptosis. Biochimica et Biophysica Acta (BBA) -Molecular Cell Research 1995, 1269, 187-199.
  105. Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004, 60, 2126-32.
  106. Molecular Operating Environment (MOE); version 2008.11; Chemical Computing Group Inc.; Montreal, Canada
  107. Englert, L.; Biela, A.; Zayed, M.; Heine, A.; Hangauer, D.; Klebe, G. Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S'-pocket of thermolysin. Biochim Biophys Acta 1800, 1192-202.
  108. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-21.
  109. Hajduk, P. J. Puzzling through fragment-based drug design. Nat Chem Biol 2006, 2, 658-9.
  110. Case, D. A.; Cheatham, T. E., 3rd; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. The Amber biomolecular simulation programs. J Comput Chem 2005, 26, 1668-88. 127. Vogtherr, M.; Saxena, K.; Hoelder, S.; Grimme, S.; Betz, M.; Schieborr, U.; Pescatore, B.; Robin, M.; Delarbre, L.; Langer, T.; Wendt, K. U.; Schwalbe, H. NMR characterization of kinase p38 dynamics in free and ligand-bound forms. Angew Chem Int Ed Engl 2006, 45, 993-7.
  111. Madhusudan; Trafny, E. A.; Xuong, N. H.; Adams, J. A.; Ten Eyck, L. F.; Taylor, S. S.; Sowadski, J. M. cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. Protein Sci 1994, 3, 176-87.
  112. Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K.; Sproesser, K.; Li, L.; Smalley, K. S.; Fong, D.; Zhu, Y. L.; Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul, C.; Shellooe, R.; Cantwell, J.; Kim, S. H.; Schlessinger, J.; Zhang, K. Y.; West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth, P.; Artis, D. R.; Herlyn, M.; Bollag, G. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A 2008, 105, 3041-6. 38. Wyatt, P. G.; Woodhead, A. J.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D. M.; Davis, D. J.; Devine, L. A.; Early, T. R.; Feltell, R. E.; Lewis, E. J.; McMenamin, R. L.; Navarro, E. F.; O'Brien, M. A.; O'Reilly, M.; Reule, M.; Saxty, G.; Seavers, L. C.; Smith, D. M.; Squires, M. S.; Trewartha, G.; Walker, M. T.; Woolford, A. J. Identification of N-(4-piperidinyl)-4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3- carboxamide (AT7519), a novel cyclin dependent kinase inhibitor using fragment-based X-ray crystallography and structure based drug design. J Med Chem 2008, 51, 4986-99.
  113. Andreeva, N. S.; Rumsh, L. D. Analysis of crystal structures of aspartic proteinases: on the role of amino acid residues adjacent to the catalytic site of pepsin- like enzymes. Protein Sci 2001, 10, 2439-50.
  114. J.; Bremer, E. Synthesis and uptake of the compatible solutes ectoine and 5- hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses.
  115. Erskine, P.; Cooper, J. The catalytic mechanism of an aspartic proteinase explored with neutron and X-ray diffraction. J Am Chem Soc 2008, 130, 7235-7.
  116. Decher, N.; Streit, A. K.; Rapedius, M.; Netter, M. F.; Marzian, S.; Ehling, P.; Schlichthorl, G.; Craan, T.; Renigunta, V.; Kohler, A.; Dodel, R. C.; Navarro-Polanco, R. A.; Preisig-Muller, R.; Klebe, G.; Budde, T.; Baukrowitz, T.; Daut, J. RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels. EMBO J 29, 2101-13.
  117. Prabhu, J.; Schauwecker, F.; Grammel, N.; Keller, U.; Bernhard, M. Functional expression of the ectoine hydroxylase gene (thpD) from Streptomyces chrysomallus in Halomonas elongata. Appl Environ Microbiol 2004, 70, 3130-2.
  118. Irwin, J. J.; Shoichet, B. K. ZINC--a free database of commercially available compounds for virtual screening. J Chem Inf Model 2005, 45, 177-82. 137. qiagen. 138. Cummings, M. D.; Farnum, M. A.; Nelen, M. I. Universal Screening Methods and Applications of ThermoFluor®. Journal of Biomolecular Screening 2006, 11, 854-863.
  119. Baum, B.; Muley, L.; Heine, A.; Smolinski, M.; Hangauer, D.; Klebe, G. Think twice: understanding the high potency of bis(phenyl)methane inhibitors of thrombin. J Mol Biol 2009, 391, 552-64.
  120. Taylor, S. S.; Yang, J.; Wu, J.; Haste, N. M.; Radzio-Andzelm, E.; Anand, G. PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta 2004, 1697, 259-69.

* Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten