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Licensed Unlicensed Requires Authentication Published by De Gruyter July 8, 2014

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues

Anastasia Tziridis, Daniel Rauh, Piotr Neumann, Petr Kolenko, Anja Menzel, Ulrike Bräuer, Christian Ursel, Peter Steinmetzer, Jörg Stürzebecher, Andrea Schweinitz, Torsten Steinmetzer and Milton T. Stubbs
From the journal Biological Chemistry


A high-resolution crystallographic structure determination of a protein–ligand complex is generally accepted as the ‘gold standard’ for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein–ligand interactions and emphasize the difficulties in predicting affinity from structure.

Dedicated to the memory of our friend and colleague Professor Dr. Jörg Stürzebecher, whose untimely death was a tragic loss to us and to the scientific community.

Corresponding author: Milton T. Stubbs, Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle/Saale, Germany, e-mail:
aPresent address: Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, D-65926 Frankfurt/Main, Germany.bPresent address: Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.cPresent address: Institut für Mikrobiologie und Genetik, Georg-August-Universität, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.dPresent address: Institute of Macromolecular Chemistry, Heyrovského nám. 2, CZ-162 06 Praha 6, Czech Republic.ePresent address: Institut für Rechtsmedizin, Otto-von-Guerike-Universität, Leipziger Str. 44, D-39120 Magdeburg, Germany.fPresent address: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.gPresent address: Institut für Physiologie II, Universitätsklinikum Jena, Kollegiengasse 9, D-07743 Jena, Germany.hPresent address: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032 Marburg, Germany.


We thank Diana Lieber for assistance with protein preparation and crystallization. Hans-Dieter Gerber (Marburg) and Curacyte Chemistry GmbH (Jena) kindly provided additional inhibitors. We also thank Gerhard Klebe (Marburg) and Norbert Sträter (Leipzig) for use of their X-ray facilities during the initial stages of this project. The diffraction data for structures TripleSer217Ile227.A1 and TripleGlu217Phe227.F1 variants were measured on the MPG/GBF beamline BW6 at DESY, while the diffraction experiments for TripleSer217Ile227.A4 and TripleGlu217Phe227.B2 variants were carried out at the Protein Structure Factory beamline BL14.1 of BESSY and Free University Berlin at BESSY. This work was supported in part by the DFG Graduiertenkolleg 1026 ‘Conformational transitions in macromolecular interactions’ to MTS.


Arnaiz, D.O., Zhao, Z., Liang, A., Trinh, L., Witlow, M., Koovakkat, S.K., and Shaw, K.J. (2000). Design, synthesis, and in vitro biological activity of indole-based factor Xa inhibitors. Bioorg. Med. Chem. Lett. 10, 957–961.10.1016/S0960-894X(00)00138-4Search in Google Scholar

Bailey, S. (1994). The CCP4 Suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763.10.1107/S0907444994003112Search in Google Scholar

Brandstetter, H., Kühne, A., Bode, W., Huber, R., von der Saal, W., Wirthensohn, K., and Engh, R.A. (1996). X-ray structure of active site-inhibited clotting factor Xa – implications for drug design and substrate recognition. J. Biol. Chem. 271, 29988–29992.10.1074/jbc.271.47.29988Search in Google Scholar

Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921.10.1107/S0907444998003254Search in Google Scholar

Dixon, M. (1972). Graphical determination of Km and Ki. Biochem. J. 129, 197.10.1042/bj1290197Search in Google Scholar

Dullweber, F., Stubbs, M.T., Musil, D., Stürzebecher, J., and Klebe, G. (2001). Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition. J. Mol. Biol. 313, 593–614.10.1006/jmbi.2001.5062Search in Google Scholar

Emsley, P. and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.10.1107/S0907444904019158Search in Google Scholar

Faull, A.W., Mayo, C.M., Preston, J., and Stocker, A. (1996). Aminoheterocyclic derivatives as antithrombotic or anticoagulant agents. (Patent, WO, 9610022).Search in Google Scholar

Feixas, F., Lindert, S., Sinko, W., and McCammon, J.A. (2014). Exploring the role of receptor flexibility in structure-based drug discovery. Biophys. Chem. 186, 31–45.10.1016/j.bpc.2013.10.007Search in Google Scholar

Hedstrom, L., Lin, T.Y., and Fast, W. (1996). Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. Biochemistry 35, 4515–4523.10.1021/bi951928kSearch in Google Scholar

Hirayama, F., Koshio, H., Katayama, N., Kurihara, H., Taniuchi, Y., Sato, K., Hisamichi, N., Sakai-Moritani, Y., Kawasaki, T., Matsumoto, Y., et al. (2002). The discovery of YM-60828: a potent, selective and orally-bioavailable Factor Xa inhibitor. Bioorg. Med. Chem. 10, 1509–1523.10.1016/S0968-0896(01)00418-7Search in Google Scholar

Huang, S.Y., Grinter, S.Z., and Zou, X. (2010). Scoring functions and their evaluation methods for protein–ligand docking: recent advances and future directions. Phys. Chem. Chem. Phys. 12, 12899–12908.10.1039/c0cp00151aSearch in Google Scholar

Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron-density maps and the location of errors in these models. Acta Crystallog. Sect. A 47, 110–119.10.1107/S0108767390010224Search in Google Scholar

Lin, Z. and Johnson, M.E. (1995). Proposed cation-π mediated binding by factor Xa: a novel enzymatic mechanism for molecular recognition. FEBS Lett. 370, 1–5.10.1016/0014-5793(95)00811-MSearch in Google Scholar

Martin, S.F. and Clements, J.H. (2013). Correlating structure and energetics in protein-ligand interactions: paradigms and paradoxes. Annu. Rev. Biochem. 82, 267–293.10.1146/annurev-biochem-060410-105819Search in Google Scholar

Nar, H. (2012). The role of structural information in the discovery of direct thrombin and factor Xa inhibitors. Trends Pharmacol. Sci. 33, 279–288.10.1016/ in Google Scholar

Otwinowski, Z. and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Macromol. Crystallog. A 276, 307–326.10.1016/S0076-6879(97)76066-XSearch in Google Scholar

Perzborn, E., Roehrig, S., Straub, A., Kubitza, D., and Misselwitz, F. (2011). The discovery and development of rivaroxaban, an oral, direct factor Xa inhibitor. Nat. Rev. Drug Discov. 10, 61–75.10.1038/nrd3185Search in Google Scholar

Pruitt, J.R., Pinto, D.J., Estrella, M.J., Bostrom, L.L., Knabb, R.M., Wong, P.C., Wright, M.R., and Wexler, R.R. (2000). Isoxazolines and isoxazoles as Factor Xa inhibitors. Bioorg. Med. Chem. Lett. 10, 685–689.10.1016/S0960-894X(00)00097-4Search in Google Scholar

Rauh, D., Reyda, S., Klebe, G., and Stubbs, M.T. (2002). Trypsin mutants for structure-based drug design: expression, refolding and crystallisation. Biol. Chem. 383, 1309–1314.10.1515/BC.2002.148Search in Google Scholar

Rauh, D., Klebe, G., Stürzebecher, J., and Stubbs, M.T. (2003). ZZ made EZ: influence of inhibitor configuration on enzyme selectivity. J. Mol. Biol. 330, 761–770.10.1016/S0022-2836(03)00617-XSearch in Google Scholar

Rauh, D., Klebe, G., and Stubbs, M.T. (2004). Understanding protein-ligand interactions: the price of protein flexibility. J. Mol. Biol. 335, 1325–1341.10.1016/j.jmb.2003.11.041Search in Google Scholar

Renatus, M., Bode, W., Huber, R., Stürzebecher, J., and Stubbs, M.T. (1998). Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase. J. Med. Chem. 41, 5445–5456.10.1021/jm981068gSearch in Google Scholar

Reyda, S., Sohn, C., Klebe, G., Rall, K., Ullmann, D., Jakubke, H.D., and Stubbs, M.T. (2003). Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity. J. Mol. Biol. 325, 963–977.10.1016/S0022-2836(02)01337-2Search in Google Scholar

Rose, P.W., Bi, C., Bluhm, W.F., Christie, C.H., Dimitropoulos, D., Dutta, S., Green, R.K., Goodsell, D.S., Prlic, A., Quesada, M., et al. (2013). The RCSB Protein Data Bank: new resources for research and education. Nucleic Acids Res. 41, D475–D482.Search in Google Scholar

Salonen, L.M., Bucher, C., Banner, D.W., Haap, W., Mary, J.L., Benz, J., Kuster, O., Seiler, P., Schweizer, W.B., and Diederich, F. (2009). Cation-π interactions at the active site of factor Xa: dramatic enhancement upon stepwise N-alkylation of ammonium ions. Angew. Chem. Int. Ed. 48, 811–814.10.1002/anie.200804695Search in Google Scholar

Salonen, L.M., Holland, M.C., Kaib, P.S., Haap, W., Benz, J., Mary, J.L., Kuster, O., Schweizer, W.B., Banner, D.W., and Diederich, F. (2012). Molecular recognition at the active site of factor Xa: cation-pi interactions, stacking on planar peptide surfaces, and replacement of structural water. Chemistry 18, 213–222.10.1002/chem.201102571Search in Google Scholar

Schärer, K., Morgenthaler, M., Paulini, R., Obst-Sander, U., Banner, D.W., Schlatter, D., Benz, J., Stihle, M., and Diederich, F. (2005). Quantification of cation-pi interactions in protein-ligand complexes: crystal-structure analysis of Factor Xa bound to a quaternary ammonium ion ligand. Angew. Chem Int. Ed. 44, 4400–4404.10.1002/anie.200500883Search in Google Scholar

Schweinitz, A., Stürzebecher, A., Stürzebecher, U., Schuster, O., Stürzebecher, J., and Steinmetzer, T. (2006). New substrate analogue inhibitors of factor Xa containing 4-amidinobenzylamide as P1 residue: part 1. Med. Chem. 2, 349–361.10.2174/157340606777724040Search in Google Scholar

Shotton, D.M. and Hartley, B.S. (1970). Amino-acid sequence of porcine pancreatic elastase and its homologies with other serine proteinases. Nature 225, 802–806.10.1038/225802a0Search in Google Scholar

Sliwoski, G., Kothiwale, S., Meiler, J., and Lowe, E.W., Jr. (2014). Computational methods in drug discovery. Pharmacol. Rev. 66, 334–395.10.1124/pr.112.007336Search in Google Scholar

Steinberg, B.A. and Becker, R.C. (2013). Structure-function relationships of factor Xa inhibitors: implications for the practicing clinician. J Thromb. Thrombolysis. 37, 234–241.10.1007/s11239-013-0991-zSearch in Google Scholar

Straub, A., Roehrig, S., and Hillisch, A. (2011). Oral, direct thrombin and factor Xa inhibitors: the replacement for warfarin, leeches, and pig intestines? Angew. Chem. Int. Ed. 50, 4574–4590.10.1002/anie.201004575Search in Google Scholar

Stubbs, M.T., Huber, R., and Bode, W. (1995). Crystal-structures of factor Xa specific inhibitors in complex with trypsin – structural grounds for inhibition of factor Xa and selectivity against thrombin. FEBS Lett. 375, 103–107.10.1016/0014-5793(95)01190-PSearch in Google Scholar

Stubbs, M.T., Reyda, S., Dullweber, F., Möller, M., Klebe, G., Dorsch, D., Mederski, W.W.K.R., and Wurziger, H. (2002). pH-dependent binding modes observed in trypsin crystals: lessons for structure-based drug design. Chembiochem 3, 246–249.10.1002/1439-7633(20020301)3:2/3<246::AID-CBIC246>3.0.CO;2-#Search in Google Scholar

Stürzebecher, J., Stürzebecher, U., Vieweg, H., Wagner, G., Hauptmann, J., and Markwardt, F. (1989). Synthetic inhibitors of bovine factor Xa and thrombin comparison of their anticoagulant efficiency. Thromb. Res. 54, 245–252.10.1016/0049-3848(89)90232-6Search in Google Scholar

Stürzebecher, J., Prasa, D., Hauptmann, J., Vieweg, H., and Wikström, P. (1997). Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine. J. Med. Chem. 40, 3091–3099.10.1021/jm960668hSearch in Google Scholar

Stürzebecher, A., Dönnecke, D., Schweinitz, A., Schuster, O., Steinmetzer, P., Stürzebecher, U., Kotthaus, J., Clement, B., Stürzebecher, J., and Steinmetzer, T. (2007). Highly potent and selective substrate analogue factor Xa inhibitors containing D-homophenylalanine analogues as P3 residue: part 2. ChemMedChem. 2, 1043–1053.10.1002/cmdc.200700031Search in Google Scholar

Teague, S.J. (2003). Implications of protein flexibility for drug discovery. Nat. Rev. Drug Discov. 2, 527–541.10.1038/nrd1129Search in Google Scholar

Turk, D., Stürzebecher, J., and Bode, W. (1991). Geometry of binding of the Nα-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino phenylalanine to thrombin and trypsin. X-ray crystal structures of their trypsin complexes and modeling of their thrombin complexes. FEBS Lett. 287, 133–138.10.1016/0014-5793(91)80033-YSearch in Google Scholar

Weiner, M.P., Costa, G.L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J.C. (1994). Site-directed mutagenesis of double-stranded DNA by the polymerase chain-reaction. Gene 151, 119–123.10.1016/0378-1119(94)90641-6Search in Google Scholar

Whitlow, M., Arnaiz, D.O., Buckman, B.O., Davey, D.D., Griedel, B., Guilford, W.J., Koovakkat, S.K., Liang, A., Mohan, R., Phillips, G.B., et al. (1999). Crystallographic analysis of potent and selective factor Xa inhibitors complexed to bovine trypsin. Acta Crystallogr. D Biol. Crystallogr. 55, 1395–1404.10.1107/S0907444999007350Search in Google Scholar

Yeh, C.H., Fredenburgh, J.C., and Weitz, J.I. (2012). Oral direct factor Xa inhibitors. Circ. Res. 111, 1069–1078.10.1161/CIRCRESAHA.112.276741Search in Google Scholar

Supplemental Material: The online version of this article (DOI 10.1515/hsz-2014-0158) offers supplementary material, available to authorized users.

Received: 2014-3-3
Accepted: 2014-4-24
Published Online: 2014-7-8
Published in Print: 2014-7-1

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