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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

Abstract

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.

Acknowledgments

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.

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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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