Merging the Binding Sites of Aldose and Aldehyde Reductase for Detection of Inhibitor Selectivity-determining Features Holger Steuber 1 , Andreas Heine 1 , Alberto Podjarny 2 and Gerhard Klebe 1 ⁎ 1 Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany 2 Département de Biologie Structurale et Génomique, IGBMC, CNRS INSERM ULP, 1 Rue Laurent Fries, 67404 Illkirch, France Received 21 November 2007; received in revised form 1 March 2008; accepted 25 March 2008 Available online 8 April 2008 Inhibition of human aldose reductase (ALR2) evolved as a promising thera- peutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein–ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermody- namics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding. © 2008 Elsevier Ltd. All rights reserved. Edited by R. Huber Keywords: selectivity-determining features; aldose reductase; inhibitor; drug design; protein–ligand interaction Introduction The development of potent drug candidates with sufficient selectivity for a given target is one of the major goals of modern drug discovery. Suitable lead compounds have to discriminate between the bind- ing pocket of the desired target and related isoforms possessing similar binding properties. Nature often achieves the required selectivity simply because the biochemical processes are evolved in well-defined physiological compartments, separated from other sites that may host putative isoforms. The situation is quite different with respect to orally administered therapeutic xenobiotics, because they flood the whole body and are distributed throughout the organism. Thus, drug candidates have to be opti- mized with respect to maximum potency, and for the required target selectivity to minimize undesired side effects. Therefore, insights into the principles determining target selectivity are of the utmost im- portance. 1 However, even in cases where structural information about the target and putative competi- tive isoforms is available, it is still a major challenge to endow a ligand with sufficient selectivity, parti- cularly if the binding site performs pronounced adaptations upon ligand accommodation. 2 Irritat- ingly enough, even slight deviations of the binding *Corresponding author. E-mail address: klebe@staff.uni-marburg.de. Abbreviations used: ALR1, aldehyde reductase; ALR2, aldose reductase; DM, double mutant; ITC, isothermal titration calorimetry; WT, wild type. doi:10.1016/j.jmb.2008.03.063 J. Mol. Biol. (2008) 379, 991–1016 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.