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

Anastasia Tziridis 1 , Daniel Rauh 1 , Piotr Neumann 1 , Petr Kolenko 1 , Anja Menzel 1 , Ulrike Bräuer 1 , Christian Ursel 1 , Peter Steinmetzer 2 , Jörg Stürzebecher 2 , Andrea Schweinitz 3 , Torsten Steinmetzer 3  and Milton T. Stubbs 1
  • 1 Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle/Saale, Germany
  • 2 Institut für Vaskuläre Medizin und Biologie, Friedrich-Schiller-Universität Jena, Nordhäuser Straße 78, D-99089 Erfurt, Germany
  • 3 Curacyte Chemistry GmbH, Winzerlaer Str. 2, D-07745 Jena, Germany
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


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.

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