Structural and thermodynamic characterization of inhibitor binding to aldose reductase: Insights into binding modes, driving forces and selectivity determinants

The TIM-barrel folded enzyme Aldose reductase (ALR2) is a valuable model system to study structural and thermodynamic features of inhibitor binding and, furthermore, represents an excellent drug target. To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the d...

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1. Verfasser: Steuber, Holger
Beteiligte: Klebe, Gerhard (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2007
Pharmazeutische Chemie
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Zusammenfassung:The TIM-barrel folded enzyme Aldose reductase (ALR2) is a valuable model system to study structural and thermodynamic features of inhibitor binding and, furthermore, represents an excellent drug target. To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Its attraction as a test system consists furthermore in the high mobility and adaptivity properties of its active site residues, giving rise to various distinct binding pocket conformers and pronounced induced-fit adaptations upon ligand binding. In chapter 2, we combine a structural characterization of the experimental binding modes observed for two virtual screening hits with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding. The nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a strongly favourable contribution to binding enthalpy in case the inhibitor is equipped with a nitro group at the corresponding position. As these data suggest, the nitro group provokes the enthalpic contribution, in addition to the H-bond mentioned above, by accepting two “non-classical” H-bonds donated by the aromatic tyrosine side chain. In chapter 3, we report on the crystal structures of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase. The inhibitor occupies with its pyridazinone head group the catalytic site whereas the chloro-benzofurane moiety penetrates into the opened specificity pocket. The high resolution structure provides some evidence that the pyridazinone group binds in a negatively charged deprotonated state whereas the neighboring His 110 residue most likely adopts a neutral uncharged state. In chapter 4, we probed the ALR2 binding site with a novel structural class of inhibitors in order to identify putative pocket adaptations. We elucidated two ALR2 crystal structures, each complexed with a member of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat, both inhibitors leave the specificity pocket in closed state. Unexpectedly, the more potent ligand extends the catalytic pocket by opening of a novel subpocket. The second studied inhibitor differs from the first only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, its binding mode differs dramatically from that of the first inhibitor. The two ligand complexes represent an impressive example, how the slight change of a chemically extended side chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships. In chapter 5, we study the binding process of inhibitors to ALR2 with respect to changes of the protonation inventory upon complex formation. As the protonation event will strongly contribute to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and -releasing functional groups of the system is of utmost importance. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr 48) as likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to remarkable extent if only the cofactor NADP+ is bound. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects. In chapter 6, the ALR2 binding site is probed for selectivity determining features, which make binding of certain ligands to ALR2 more attractive than to the concurrent isoform aldehyde reductase (ALR1). The resulting mutational constructs of ALR2 are probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and ITC. Accurate crystal structure-determination of protein-ligand complexes is the starting point for further design hypotheses to predict novel leads with improved properties. This widely accepted practise relies on the assumption that the crystal structure of a given protein-ligand complex is unique and independent of the protocol applied to produce the crystals. In chapter 7, we present two examples indicating that this assumption is not generally given.
DOI:https://doi.org/10.17192/z2007.0474