Publikationsserver der Universitätsbibliothek Marburg

Titel:Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin
Autor:Cramer, Jonathan
Weitere Beteiligte: Klebe, Gerhard (Prof. Dr.)
URN: urn:nbn:de:hebis:04-z2017-04658
DDC: Chemie
Titel (trans.):Inhibitor Synthese und biophysikalische Charakterisierung von Protein–Ligand–Solvens Interaktionen Eine Analyse der Thermodynamik und Kinetik der Ligandenbindung an Thermolysin


Thermodynamics, Synthese, Kinetics, Thermodynamik, Synthesis, Bindungskinetik, Phosphonamidat, Thermolysin, Kinetik, Chemische Synthese, ITC, Thermodynamik, Wirkstoff, SPR, Proteine, Phosphonamidate, Thermolysin

In the pre-clinical development stages of most drug design campaigns, the equilibrium binding affinity of a prospective lead candidate, in the form of an IC50, Kd or ΔG° value, is the most commonly employed benchmark parameter for its effectiveness as a putative drug. Hydrogen bonding, van der Waals and electrostatic interactions, as well as hydrophobic effects are among the most prominent factors that contribute to binding. In structure based design approaches, these interactions can routinely be linked to a structural motif of a drug molecule, which can greatly assist in the construction of compounds with a desired set of properties. Equilibrium binding affinity can also be expressed in terms of kinetics, were the steady-state constant Kd is defined as the ratio of the rate constants of dissociation (kd) and association (ka). The thermodynamic expression ΔG° can be subdivided into an enthalpic (ΔH°) and an entropic (–TΔS°) term. In either case, the molecular mechanisms that define the kinetics of binding or the compensation of enthalpic and entropic contributions are not fully understood. The goal of this dissertation is the in-depth investigation of the molecular processes that drive protein–ligand interactions. A special focus is set on the partitioning of thermodynamic and kinetic parameters into their respective microscopic elements. For this, the metalloprotease thermolysin (TLN) is used as a model system. This protein is well characterized and represents a robust system with excellent crystallographic properties and a thoroughly documented inhibitor class. The first publication (Chapter 2) presents an improved strategy for the synthesis and purification of phosphonamidate peptides that are known as potent inhibitors of TLN. Due to the inherent instability of the phosphorous–nitrogen bond, the introduction of polar functional groups into the inhibitor scaffold is quite challenging. Here, a synthetic strategy is presented that minimizes the amount of hydrolysis during peptide coupling, deprotection and purification through the use of an allyl-based protection system and a solid-phase extraction (SPE) protocol for the final purification step. This allows the synthesis of highly pure TLN inhibitors incorporating a variety of functional groups for use in biophysical experiments. In the second publication (Chapter 3), a strategy for the design of inhibitors is highlighted, which relies on the targeted design of water networks that are formed around a protein–ligand complex. Based on information from a previous study, the shape of a hydrophobic portion of a TLN ligand is altered in a way that allows a beneficial stabilization of water molecules in the first solvation layer of the complex. Supported by molecular dynamics simulations, a series of diastereomeric inhibitors is synthesized and the binding process is characterized by X-ray crystallography, isothermal titration calorimetry (ITC) and surface plasmon resonance spectroscopy (SPR). The optimization of the hydrophobic P2’ moiety results in a 50-fold affinity enhancement compared to the original methyl substituted ligand. This improvement is mainly driven by a favorable enthalpic term that originates from the stabilization of water polygons in the solvation shell. In the follow-up study in Chapter 4, the binding signature of a series of inhibitors that place a charged and polar moiety in the solvent exposed S2’ pocket of TLN is investigated. Here, a partially hydrated ammonium group is gradually retracted deeper into the hydrophobic protein environment. From the crystal structures it is evident that the polar ligands do not recruit an increased amount of water molecules into their solvation layer when compared to related analogues that feature a purely aliphatic residue at the solvent interface. The penalty for the partial desolvation of the charged functional group, in combination with the lack of a strongly ordered water network, results in a severe affinity decrease that is driven by an unfavorable enthalpic term. The deep, hydrophobic S1’ pocket of TLN determines the substrate specificity of the protease and is commonly addressed by high affinity inhibitors. Experimental evidence from previous studies suggests, however, that this apolar crevice is only poorly solvated in the absence of an interaction partner. With the study in Chapter 5, an attempt for the experimental analysis of the hydration state of the S1’ pocket is presented. For this, a special inhibitor is designed that transforms the protein pocket into a cavity, while simultaneously providing enough empty space for the accommodation of several water molecules. A detailed analysis of an experimentally phased electron density map reveals that the cavity remains completely unsolvated and thus, vacuous. As an intriguing prospect for the exploitation of such poorly hydrated protein pockets in drug design, the placement of an iso-pentyl moiety in the ligand’s P1’ position results in a dramatic, enthalpically driven gain in affinity by a factor of 41 000. With a detailed structural analysis of a series of chemically diverse TLN inhibitors, the kinetics of the protein–ligand binding process are investigated in Chapter 6. From the SPR derived kinetic information, it becomes apparent that the nature of the functional group in the P2’ position of a thermolysin inhibitor has a significant impact on its dissociation kinetics. This property can be linked to the interaction between the respective functionality of a ligand and Asn112, a residue that lines the active site of the protease and is commonly believed to align a substrate for proteolytic cleavage. This residue undergoes a significant conformational change when the protein transitions from its closed state to its open form, from which a ligand is released. Interference with this retrograde induced-fit mechanism through strong hydrogen-bonding interactions to an inhibitor results in a pronounced deceleration of the dissociation process. The case of the known inhibitor ZFPLA demonstrates that a further restriction of the rotation of Asn112 by a steric barrier in the P1 position of a ligand, can reduce the rate constant of dissociation by a factor of 74 000. Fragment-based lead discovery has become a popular method for the generation of prospective drug molecules. The weak affinity of fragments and the necessity for high concentrations, however, can result in false-positive signals from the initial binding assays that routinely plague fragment-based screening. The pursuit of such a “red herring” can lead to a significant loss of time and resources. In Chapter 7, a molecule that emerged as one of the most potent binders from an elaborate fragment screen against the aspartic protease endothiapepsin is identified as a false-positive. Detailed crystallographic, HPLC and MS experiments reveal that the affinity detected in multiple assays can in fact be attributed to another compound. This entity is formed from the initially employed molecule in a reaction cascade that results in a major rearrangement of its heterocyclic core structure. Supported by quantum chemical calculations and NMR experiments, a mechanism for the formation of the elusive compound is proposed and its binding mode analyzed by X-ray crystallography.

Bibliographie / References

  1. Qvist, J., Davidovic, M., Hamelberg, D., and Halle, B. (2008) A dry ligand-binding cavity in a solvated protein. Proc. Natl. Acad. Sci. USA, 105, 6296-6301.
  2. Vallee, M.R.J., Artner, L.M., Dernedde, J., and Hackenberger, C.P.R. (2013) Alkyne phosphonites for sequential azide-azide couplings. Angew. Chem., Int. Ed. Engl., 52, 9504-9508.
  3. Bissantz, C., Kuhn, B., and Stahl, M. (2010) A medicinal chemist's guide to molecular interactions. J.
  4. Yasukawa, K., Kusano, M., and Inouye, K. (2007) A new method for the extracellular production of 263.
  5. Gao, Y., He, X.-Y., Wang, Z.-B., et al. (2009) A new type of pseudorotaxanes based on cucurbit[6]uril and bis-cyanopyridyl alkane compounds. Supramol. Chem., 21, 699-706.
  6. Petrillo, E.W.J., and Ondetti, M.A. (1982) Angiotensin-converting enzyme inhibitors: medicinal chemistry and biological actions. Med. Res. Rev., 2, 1-41.
  7. McMurray, J.J. V, Packer, M., Desai, A.S., et al. (2014) Angiotensin-neprilysin inhibition versus enalapril in heart failure. N. Engl. J. Med., 371, 993-1004.
  8. Thorn, A., and Sheldrick, G.M. (2011) ANODE: Anomalous and heavy-atom density calculation. J.
  9. Mucha, A., Kunert, A., Grembecka, J., et al. (2006) A phosphonamidate containing aromatic Nterminal amino group as inhibitor of leucine aminopeptidase-design, synthesis and stability. Eur. J.
  10. Matthews, B.W., and Liu, L. (2009) A review about nothing: Are apolar cavities in proteins really empty? Protein Sci., 18, 494-502.
  11. Li, A.J.-J., and Nussinov, R. (1998) A set of van der Waals and Coulombic radii of protein atoms for molecular and solvent-accessible surface calculation, packing evaluation, and docking. Proteins Struct. Funct. Genet., 32, 111-127.
  12. Sheldrick, G.M. (2007) A short history of SHELX. Acta Crystallogr. Sect. A Found. Crystallogr., 64, 112-122.
  13. Luckner, S.R., Liu, N., Am Ende, C.W., et al. (2010) A Slow, Tight Binding Inhibitor of InhA, the Enoyl-Acyl Carrier Protein Reductase from Mycobacterium tuberculosis. J. Biol. Chem., 285, 14330- 14337.
  14. Koster, H., Craan, T., Brass, S., et al. (2011) A small nonrule of 3 compatible fragment library provides high hit rate of endothiapepsin crystal structures with various fragment chemotypes. J. Med. Chem., 54, 7784-7796.
  15. Thomas, S.P., and Aggarwal, V.K. (2009) Asymmetric Hydroboration of 1,1-Disubstituted Alkenes.
  16. Freire, E. (2009) A thermodynamic approach to the affinity optimization of drug candidates. Chem.
  17. Southall, N.T., Dill, K.A., and Haymet, A.D.J. (2002) A View of the Hydrophobic Effect. J. Phys.
  18. Chaires, J.B. (2008) Calorimetry and thermodynamics in drug design. Annu. Rev. Biophys., 37, 135- 151.
  19. Sonavane, S., and Chakrabarti, P. (2008) Cavities and Atomic Packing in Protein Structures and Interfaces. PLoS Comput. Biol., 4, e1000188.
  20. Lee, C., Maeng, J.S., Kocher, J.P., et al. (2001) Cavities of alpha(1)-antitrypsin that play structural and functional roles. Protein Sci., 10, 1446-1453.
  21. Voss, N.R., and Gerstein, M. (2010) 3V: Cavity, channel and cleft volume calculator and extractor.
  22. Jt. CCP4 ESF-EACBM Newsl. Protein Crystallogr., 31, 34-38.
  23. Schiebel, J., Gaspari, R., Sandner, A., et al. (2017) Charges Shift Protonation: Ultimately, Neutron Diffraction Discloses that Aniline and 2-Aminopyridine Become Protonated Upon Binding to Trypsin. Angew. Chemie - Int. Ed.
  24. Vallee, M.R.J., Majkut, P., Krause, D., et al. (2015) Chemoselective bioconjugation of triazole phosphonites in aqueous media. Chem. Eur. J., 21, 970-974.
  25. Pearlstein, R.A., Sherman, W., and Abel, R. (2013) Contributions of water transfer energy to proteinligand association and dissociation barriers: Watermap analysis of a series of p38a MAP kinase inhibitors. Proteins Struct. Funct. Bioinforma., 81, 1509-1526.
  26. Martin, S.F., and Clements, J.H. (2013) Correlating structure and energetics in protein-ligand interactions: paradigms and paradoxes. Annu. Rev. Biochem., 82, 267-93.
  27. Ernst, J.A., Clubb, R.T., Zhou, H.X., et al. (1995) Demonstration of positionally disordered water within a protein hydrophobic cavity by NMR. Science, 267, 1813-1817.
  28. Whitesides, G.M., and Krishnamurthy, V.M. (2005) Designing ligands to bind proteins. Q. Rev.
  29. Morgan, B., Scholtz, J.M., Ballinger, M.D., et al. (1991) Differential binding energy: A detailed evaluation of the influence of hydrogen-bonding and hydrophobic groups on the inhibition of thermolysin by phosphorus-containing inhibitors. J. Am. Chem. Soc., 113, 297-307.
  30. Leavitt, S., and Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol., 11, 560-566.
  31. Yu, B., Blaber, M., Gronenborn, A.M., et al. (1999) Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography. Proc. Natl. Acad. Sci. USA, 96, 103-108.
  32. Biela, A., Nasief, N.N., Betz, M., et al. (2013) Dissecting the Hydrophobic Effect on the Molecular Level: The Role of Water, Enthalpy, and Entropy in Ligand Binding to Thermolysin. Angew. Chemie 94.
  33. Cowtan, K. (1994) “dm”: an automated procedure for phase improvement by density modification.
  34. Freire, E. (2008) Do enthalpy and entropy distinguish first in class from best in class? Drug Discov.
  35. Parr, R.G., Szentpály, L. v., and Liu, S. (1999) Electrophilicity Index. J. Am. Chem. Soc., 121, 1922- 1924.
  36. Zhu, S., and Buchwald, S.L. (2014) Enantioselective CuH-Catalyzed Anti-Markovnikov Hydroamination of 1,1-Disubstituted Alkenes. J. Am. Chem. Soc., 136, 15913-15916.
  37. Funalot, B., Ouimet, T., Claperon, A., et al. (2004) Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer's disease. Mol. Psychiatry, 9, 1059, 1122-1128.
  38. Kellis, J.T., Nyberg, K., and Fersht, A.R. (1989) Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry, 28, 4914-4922.
  39. Gallicchio, E., Kubo, M.M., and Levy, R.M. (1998) Entropy-Enthalpy Compensation in Solvation and Ligand Binding Revisited. J. Am. Chem. Soc., 120, 4526-4527.
  40. Chodera, J.D., and Mobley, D.L. (2013) Entropy-enthalpy compensation: role and ramifications in biomolecular ligand recognition and design. Annu. Rev. Biophys., 42, 121-42.
  41. Carroll, M.J., Mauldin, R. V, Gromova, A. V, et al. (2012) Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat. Chem. Biol., 8, 246-252.
  42. English, A.C., Groom, C.R., and Hubbard, R.E. (2001) Experimental and computational mapping of the binding surface of a crystalline protein. Protein Eng., 14, 47-59.
  43. Prangé, T., Schiltz, M., Pernot, L., et al. (1998) Exploring hydrophobic sites in proteins with xenon or krypton. Proteins Struct. Funct. Genet., 30, 61-73.
  44. Kamysz, W., Okroj, M., Lempicka, E., et al. (2004) Fast and efficient purification of synthetic peptides by solid-phase extraction. Acta Chromatogr., 14, 180-186.
  45. Neudert, G., and Klebe, G. (2011) fconv: format conversion, manipulation and feature computation of molecular data. Bioinformatics, 27, 1021-1022.
  46. Simeonov, A., Jadhav, A., Thomas, C.J., et al. (2008) Fluorescence Spectroscopic Profiling of Compound Libraries. J. Med. Chem., 51, 2363-2371.
  47. Roden, L.D., and Myszka, D.G. (1996) Global Analysis of a Macromolecular Interaction Measured on BIAcore. Biochem. Biophys. Res. Commun., 225, 1073-1077.
  48. Liu, Y., Stoll, V.S., Richardson, P.L., et al. (2004) Hepatitis C NS3 protease inhibition by peptidylalpha-ketoamide inhibitors: kinetic mechanism and structure. Arch. Biochem. Biophys., 421, 207- 216.
  49. Schlitz, M., Shepard, W., Fourme, R., et al. (1997) High-Pressure Krypton Gas and Statistical HeavyAtom Refinement: a Successful Combination of Tools for Macromolecular Structure Determination.
  50. Pape, T., and Schneider, T.R. (2004) HKL2MAP : a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Crystallogr., 37, 843-844.
  51. Setny, P., Baron, R., and McCammon, J.A. (2010) How Can Hydrophobic Association Be Enthalpy Driven? J Chem Theory Comput, 6, 2866-2871.
  52. Mucha, A., Grembecka, J., Cierpicki, T., and Kafarski, P. (2003) Hydrolysis of the Phosphonamidate Bond in Phosphono Dipeptide Analogues--- the Influence of the Nature of the N-Terminal Functional Group. European J. Org. Chem., 2003, 4797-4803.
  53. Winquist, J., Geschwindner, S., Xue, Y., et al. (2013) Identification of Structural−Kinetic and Structural−ermodynamic Relationships for rombin Inhibitors. Biochemistry, 52, 613-626.
  54. Gruner, S., Neeb, M., Barandun, L.J., et al. (2014) Impact of protein and ligand impurities on ITCderived protein-ligand thermodynamics. Biochim. Biophys. Acta, 1840, 2843-2850.
  55. Betz, M., Wulsdorf, T., Krimmer, S.G., and Klebe, G. (2015) Impact of Surface Water Layers on Protein-Ligand Binding: How Well Are Experimental Data Reproduced by Molecular Dynamics Simulations in a Thermolysin Test Case. J. Chem. Inf. Model., 56, 223-233.
  56. Tanwar, A.S., Goyal, V.D., Choudhary, D., et al. (2013) Importance of Hydrophobic Cavities in Allosteric Regulation of Formylglycinamide Synthetase: Insight from Xenon Trapping and Statistical Coupling Analysis. PLoS One, 8, e77781.
  57. Pauli, G.F., Chen, S.-N., Simmler, C., et al. (2014) Importance of purity evaluation and the potential of quantitative (1)H NMR as a purity assay. J. Med. Chem., 57, 9220-9231.
  58. Hubbard, S.J., Gross, K.H., and Argos, P. (1994) Intramolecular Cavities in Globular Proteins.
  59. Asada, T., Koi, Y., Arakawa, R., et al. (2014) Isolation techniques for anthocyanidin 3,5-diglucosides and their related chemicals using supramolecules technique, and two solid-phase extraction cartridges. J. Chromatogr. A, 1351, 21-28.
  60. Jelesarov, I., and Bosshard, H.R. (1999) Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to invesitigate the energetics of biomolecular recognition. J. Mol.
  61. J. Mol. Biol., 302, 955-977.
  62. Ladbury, J.E. (1996) Just add water! The effect of water on the specificity of protein-ligand binding sites and its potential application to drug design. Chem. Biol., 3, 973-980.
  63. Guo, J., Huang, W., and Scanlan, T.S. (1994) Kinetic and Mechanistic Characterization of an Efficient Hydrolytic Antibody: Evidence for the Formation of an Acyl Intermediate. J. Am. Chem. Soc., 116, 6062-6069.
  64. Biela, A., Sielaff, F., Terwesten, F., et al. (2012) Ligand binding stepwise disrupts water network in thrombin: enthalpic and entropic changes reveal classical hydrophobic effect. J. Med. Chem., 55, 6094-6110.
  65. Geschwindner, S., Ulander, J., and Johansson, P. (2015) Ligand binding thermodynamics in drug discovery: still a hot tip? J. Med. Chem., 58, 6321-6335.
  66. Wang, L., Berne, B.J., and Friesner, R.A. (2011) Ligand binding to protein-binding pockets with wet and dry regions. Proc. Natl. Acad. Sci. USA, 108, 1326-1330.
  67. Holdgate, G.A., and Ward, W.H.J. (2005) Measurements of binding thermodynamics in drug discovery. Drug Discov. Today, 10, 1543-1550.
  68. Macrae, C.F., Bruno, I.J., Chisholm, J.A., et al. (2008) Mercury CSD 2.0 -- new features for the visualization and investigation of crystal structures. J. Appl. Crystallogr., 41, 466-470.
  69. Leung, C.S., Leung, S.S.F., Tirado-Rives, J., and Jorgensen, W.L. (2012) Methyl Effects on Protein−Ligand Binding. J. Med. Chem., 55, 4489-4500.
  70. Kishore Kumar, G.D., Saenz, D., Lokesh, G.L., and Natarajan, A. (2006) Microwave-assisted cleavage of phosphate, phosphonate and phosphoramide esters. Tetrahedron Lett., 47, 6281-6284.
  71. Hummer, G. (2010) Molecular binding: Under water's influence. Nat. Chem., 2, 906-7.
  72. Pan, A.C., Borhani, D.W., Dror, R.O., and Shaw, D.E. (2013) Molecular determinants of drugreceptor binding kinetics. Drug Discov. Today, 18, 667-673.
  73. Biavardi, E., Favazza, M., Motta, A., et al. (2009) Molecular recognition on a cavitand-functionalized silicon surface. J. Am. Chem. Soc., 131, 7447-7455.
  74. Bhat, S., and Purisima, E.O. (2006) Molecular surface generation using a variable-radius solvent probe. Proteins Struct. Funct. Genet., 62, 244-261.
  75. Young, T., Abel, R., Kim, B., et al. (2007) Motifs for molecular recognition exploiting hydrophobic enclosure in protein-ligand binding. Proc. Natl. Acad. Sci. USA, 104, 808-813.
  76. Bruno, I.J., Cole, J.C., Edgington, P.R., et al. (2002) New software for searching the Cambridge Structural Database and visualizing crystal structures. Acta Crystallogr., Sect. B Struct. Sci., 58, 389- 397.
  77. Baell, J.B., and Holloway, G.A. (2010) New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem., 53, 2719-2740.
  78. Schleyer, P. von R., Maerker, C., Dransfeld, A., et al. (1996) Nucleus-Independent Chemical Shifts: A Simple and Efficient Aromaticity Probe. J. Am. Chem. Soc., 118, 6317-6318.
  79. Wolfenden, R., and Radzicka, A. (1994) On the probability of finding a water molecule in a nonpolar cavity. Science, 265, 936-937.
  80. Smollich, M., Gotte, M., Yip, G.W., et al. (2007) On the role of endothelin-converting enzyme-1 (ECE-1) and neprilysin in human breast cancer. Breast Cancer Res. Treat., 106, 361-369.
  81. Winn, M.D., Ballard, C.C., Cowtan, K.D., et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr., 67, 235-242.
  82. Karunaratne, V., and Dolphin, D. (1996) Oxidation of substituted 2-methylpyrroles with perhalogenated metalloporphyrins: A one-pot synthesis of dipyrromethanes. Tetrahedron Lett., 37, 603-604.
  83. Sonnhammer, E.L., Eddy, S.R., and Durbin, R. (1997) Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins, 28, 405-420.
  84. Adams, P.D., Afonine, P. V, Bunkoczi, G., et al. (2010) PHENIX: a comprehensive Python-based Cramer, J., Krimmer, S.G., Fridh, V., et al. (2017) Elucidating the Origin of Long Residence Time Urzhumtsev, A., Afonine, P.V., Lunin, V.Y., et al. (2014) Metrics for comparison of crystallographic maps. Acta Crystallogr. Sect. D Biol. Crystallogr., 70, 2593-2606.
  85. Mookhtiar, K.A., Marlowe, C.K., Bartlett, P.A., and van Wart, H.E. (1987) Phosphonamidate inhibitors of human neutrophil collagenase. Biochemistry, 26, 1962-1965.
  86. McLeod, D.A., Brinkworth, R.I., Ashley, J.A., et al. (1991) Phosphonamidates and phosphonamidate esters as HIV-1 protease inhibitors. Bioorg. Med. Chem. Lett., 1, 653-658.
  87. Sakurai, N., and Ohmiya, S. (1993) Photoaddition reaction of 1,2-dialkyl-indoles and -pyrroles to 1- methyl-2-pyridone via proton transfer from the 2-methylene group of the indole or pyrrole. J. Chem.
  88. Ohmiya, S., Noguchi, M., Ina, S., et al. (1992) Photoaddition Reaction of Pyrroles and Indoles to NMethyl-2-pyridone. Chem. Pharm. Bull., 40, 854-857.
  89. Oxford Cryosystems (1999) Portable xenon pressure chamber. Acta Crystallogr. Sect. D Biol.
  90. Garvey, E.P., Schwartz, B., Gartland, M.J., et al. (2009) Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant. Biochemistry, 48, 1644-1653.
  91. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr., 26, 283-291.
  92. Lang, P.T., Holton, J.M., Fraser, J.S., and Alber, T. (2014) Protein structural ensembles are revealed by redefining X-ray electron density noise. Proc. Natl. Acad. Sci. USA, 111, 237-242.
  93. Gerstein, M., Richards, F.M., Chapman, M.S., and Connolly, M.L. (2001) Protein surfaces and volumes: measurement and use, in International Tables for Crystallography Volume F: Crystallography of biological macromolecules (eds.Rossmann, M.G., and Arnold, E.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 531-545.
  94. de la Figuera Gomez, T.H., Arques, J.S., Jones, R.A., et al. (1985) Pyrrole studies. Part 32. A novel ring-cleavage reaction of the pyridazine ring during the reaction of 6H-pyrrolo[3,4-d]pyridazines with dimethyl acetylenedicarboxylate. J. Chem. Soc. Perkin Trans. 1, 899-902.
  95. Chang, A., Schiebel, J., Yu, W., et al. (2013) Rational optimization of drug-target residence time: insights from inhibitor binding to the Staphylococcus aureus FabI enzyme-product complex.
  96. Eriksson, A.E., Baase, W.A., Zhang, X.-J., et al. (1992) Response of a protein structure to cavitycreating mutations and its relation to the hydrophobic effect. Science, 255, 178-183.
  97. Vallone, B., and Brunori, M. (2004) Roles for holes: are cavities in proteins mere packing defects? Ital. J. Biochem., 53, 46-53.
  98. Krissinel, E., and Henrick, K. (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr., Sect. D Biol. Crystallogr., 60, 2256-2268.
  99. Schmidtke, P., Luque, F.J., Murray, J.B., and Barril, X. (2011) Shielded Hydrogen Bonds as Structural Determinants of Binding Kinetics: Application in Drug Design. J. Am. Chem. Soc., 133, 18903-18910.
  100. Quillin, M.L., Breyer, W.A., Griswold, I.J., and Matthews, B.W. (2000) Size versus polarizability in protein-ligand interactions: binding of noble gases within engineered cavities in phage T4 lysozyme.
  101. Holden, H.M., Tronrud, D.E., Monzingo, A.F., et al. (1987) Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry, 26, 8542-8553.
  102. Hennion, M.-C. (1999) Solid-phase extraction: Method development, sorbents, and coupling with liquid chromatography. J. Chromatogr. A, 856, 3-54.
  103. Nilsson, U.J., Fournier, E.J., and Hindsgaul, O. (1998) Solid-phase extraction on C18 silica as a purification strategy in the solution synthesis of a 1-thio-beta-D-galactopyranoside library. Bioorg.
  104. Hua, T.D., Lamaty, F., Souriau, C., et al. (1996) Specific recognition of a tetrahedral phosphonamidate transition state analogue group by a recombinant antibody Fab fragment. Amino Acids, 10, 167-172.
  105. Schneider, P., Röthlisberger, M., Reker, D., et al. (2016) Spotting and designing promiscuous ligands for drug discovery. Chem. Commun., 52, 1135-1138.
  106. Vallee, M.R.J., Majkut, P., Wilkening, I., et al. (2011) Staudinger-phosphonite reactions for the chemoselective transformation of azido-containing peptides and proteins. Org. Lett., 13, 5440-5443.
  107. Holland, D.R., Hausrath, A.C., Juers, D., and Matthews, B.W. (1995) Structural analysis of zinc substitutions in the active site of thermolysin. Protein Sci., 4, 1955-1965.
  108. Adamek, D.H., Guerrero, L., Blaber, M., and Caspar, D.L.D. (2005) Structural and energetic consequences of mutations in a solvated hydrophobic cavity. J. Mol. Biol., 346, 307-318.
  109. Xu, J., Baase, W.A., Quillin, M.L., et al. (2001) Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme. Protein Sci., 10, 1067-1078.
  110. Arolas, J.L., Botelho, T.O., Vilcinskas, A., and Gomis-Ruth, F.X. (2011) Structural evidence for standard-mechanism inhibition in metallopeptidases from a complex poised to resynthesize a peptide bond. Angew. Chem., Int. Ed. Engl., 50, 10357-10360.
  111. Kruse, A.C., Hu, J., Pan, A.C., et al. (2012) Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature, 482, 552-556.
  112. Christianson, D.W., and Lipscomb, W.N. (1986) Structure of the complex between an unexpectedly hydrolyzed phosphonamidate inhibitor and carboxypeptidase A. J. Am. Chem. Soc., 108, 545-546.
  113. Schneider, T.R., and Sheldrick, G.M. (2002) Substructure solution with SHELXD. Acta Crystallogr.
  114. Wilkening, I., del Signore, G., and Hackenberger, C.P.R. (2011) Synthesis of phosphonamidate peptides by Staudinger reactions of silylated phosphinic acids and esters. Chem. Commun. (Camb)., 47, 349-351.
  115. de Medina, P., Ingrassia, L.S., and Mulliez, M.E. (2003) Synthesis of the first stable phosphonamide transition state analogue. J. Org. Chem., 68, 8424-8430.
  116. Edink, E., Jansen, C., Leurs, R., and De Esch, I.J.P. (2010) The heat is on: Thermodynamic analysis in fragment-based drug discovery. Drug Discov. Today Technol., 7, 189-201.
  117. Mueller, U., Förster, R., Hellmig, M., et al. (2015) The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives. Eur. Phys. J. Plus, 130, 141.
  118. Matthews, B.W. (1972) The Macromolecules, 5, 818-819.
  119. Goldberg, R.N., Kishore, N., and Lennen, R.M. (2002) Thermodynamic Quantities for the Ionization Reactions of Buffers. J. Phys. Chem. Ref. Data, 31, 231-370.
  120. Ferenczy, G.G., and Keseru, G.M. (2010) Thermodynamics guided lead discovery and optimization.
  121. Krimmer, S.G., and Klebe, G. (2015) Thermodynamics of protein-ligand interactions as a reference for computational analysis: how to assess accuracy, reliability and relevance of experimental data. J.
  122. Hausrath, A.C., and Matthews, B.W. (2002) Thermolysin in the absence of substrate has an open conformation. Acta Crystallogr., Sect. D Biol. Crystallogr., 58, 1002-1007.
  123. Olsson, T.S.G., Williams, M.A., Pitt, W.R., and Ladbury, J.E. (2008) The Thermodynamics of Protein-Ligand Interaction and Solvation: Insights for Ligand Design. J. Mol. Biol., 384, 1002-1017.
  124. Adekoya, O.A., and Sylte, I. (2009) The thermolysin family (M4) of enzymes: Therapeutic and 128.
  125. Matthews, B.W., Morton, A.G., and Dahlquist, F.W. (1995) Use of NMR to Detect Water Within Nonpolar Protein Cavities. Science, 270, 1847-1849.
  126. Barratt, E., Bingham, R.J., Warner, D.J., et al. (2005) Van der Waals Interactions Dominate LigandProtein Association in a Protein Binding Site Occluded from Solvent Water. J. Am. Chem. Soc., 127, 11827-11834.
  127. Ball, P. (2008) Water as an active constituent in cell biology. Chem. Rev., 108, 74-108.
  128. Vaitheeswaran, S., Yin, H., Rasaiah, J.C., and Hummer, G. (2004) Water clusters in nonpolar cavities.
  129. Bortolato, A., Tehan, B.G., Bodnarchuk, M.S., et al. (2013) Water Network Perturbation in Ligand Binding: Adenosine A 2A Antagonists as a Case Study. J. Chem. Inf. Model., 53, 1700-1713.
  130. Breiten, B., Lockett, M.R., Sherman, W., et al. (2013) Water Networks Contribute to Enthalpy / Entropy Compensation in Protein-Ligand Binding. J. Am. Chem. Soc., 135, 15579-15584.
  131. Lee, J., and Kim, S.H. (2009) Water polygons in high-resolution protein crystal structures. Protein Sci., 18, 1370-1376.
  132. Homans, S.W. (2007) Water, water everywhere - except where it matters? Drug Discov. Today, 12, 534-539.
  133. Kleywegt, G.J., and Jones, T.A. (1996) xdlMAPMAN and xdlDATAMAN - Programs for reformatting, analysis and manipulation of biomacromolecular electron-density maps and reflection data sets. Acta Crystallogr. Sect. D Biol. Crystallogr., 52, 826-828.
  134. Biela, A., Nasief, N.N., Betz, M., et al. (2013) Zerlegung des hydrophoben Effekts auf molekularer Ebene: Die Rolle von Wasser, Enthalpie und Entropie bei der Ligandenbindung an Thermolysin.

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