Structural and thermodynamic characterization of inhibitor binding to aldose reductase: Insights into binding modes, driving forces and selectivity determinants
Steuber, Holger
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.
Philipps-Universität Marburg
Chemistry + allied sciences
urn:nbn:de:hebis:04-z2007-04747
https://doi.org/10.17192/z2007.0474
opus:1694
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
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Structural and thermodynamic characterization of inhibitor binding to aldose reductase: Insights into binding modes, driving forces and selectivity determinants
Aldose reductase
Crystal structure
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.
Pharmazeutische Chemie
2007-06-05
219
application/pdf
ths
Prof. Dr.
Klebe
Gerhard
Klebe, Gerhard (Prof. Dr.)
Strukturelle und thermodynamische Untersuchungen der Inhibitorbindung an das Enzym Aldose Reduktase zum Verständnis von Bindungsmoden, Triebkräften und selektivitätsbestimmenden Eigenschaften
Selectivity
urn:nbn:de:hebis:04-z2007-04747
Chemistry + allied sciences
Chemie
https://doi.org/10.17192/z2007.0474
Wirkstoffdesign
Steuber, Holger
Steuber
Holger
Philipps-Universität Marburg
Protein-Ligand-Wechselwirkung
Drug design
Selektivität
Kristallstruktur
doctoralThesis
Fachbereich Pharmazie
Protein ligand interaction
2011-08-10
Aldose Reduktase
monograph
2007
opus:1694
Das Enzym Aldose Reduktase (ALR2) stellt ein wertvolles Testsystem dar, um strukturelle und thermodynamische Eigenschaften der Inhibitorbindung zu charakterisieren und ist darüber hinaus ein geeignetes Zielenzym für eine Arzneistoff-Intervention. Zudem ist das Enzym ein hervorragendes Testsystem für Strategien zur Leitstrukturfindung, da es aufgrund der hohen Mobilität der Aminosäure-Reste in der Bindetasche in der Lage ist, verschiedene Konformere einzunehmen und sich ausgesprochen vielseitig an Liganden anzupassen.
Zu Beginn dieser Arbeit wird die Strukturbestimmung der Bindungsmoden von durch Virtuelles Screening identifizierten Liganden ergänzt durch Isothermale Titrationskalorimetrie (ITC), um Einblicke in die Triebkräfte des Bindungsprozesses zu erhalten. In Kapitel 3 werden die Kristallstrukturen eines neuartigen Sulfonyl-Pyridazinons im Komplex mit ALR2 beschrieben. Der Inhibitor besetzt mit seiner Pyridazinon-Kopfgruppe die katalytische Tasche, während der chlor-substituierte Benzofuran-Teil die Spezifitätstasche belegt. Die hochaufgelöste Struktur legt nahe, dass die Pyridazinon-Gruppe in deprotoniertem, negativ geladenem Zustand bindet, während das benachbarte Histidin einen ungeladenen Zustand einnimmt.
In Kapitel 4 wird die Bindetasche der ALR2 mit einer neuartigen Strukturklasse von Inhibitoren sondiert, um mögliche neue Adaptationsvorgänge des Enzyms sowie den Bindungsmodus der Liganden zu bestimmen und deren Struktur-Wirkungs-Beziehungen zu verstehen. Zwei Kristallstrukturen mit einem Liganden der kürzlich veröffentlichten Naphtho[1,2-d]isothiazol-essigsäuren wurden bestimmt. Im Gegensatz zur ursprünglichen Designhypothese zeigt sich die Spezifitätstasche in den Strukturen in geschlossenem Zustand. Der potentere der beiden Liganden erweitert die katalytische Tasche durch Öffnung einer neuen Subtasche. Der zweite Ligand unterscheidet sich vom ersten nur durch einen angefügten Glycolsäure-Teil, mit dem eine der Carboxylgruppen des Liganden verestert ist. Trotz dieser nur geringfügigen Modifikation unterscheidet sich der Bindungsmodus in dramatischer Weise von dem des zuerst untersuchten Liganden. In Kapitel 5 wird der Bindungsprozess von Inhibitoren an ALR2 im Hinblick auf Änderungen von Protonierungszuständen untersucht. Die durchgeführten ITC-Messungen legen eine Protonenaufnahme bei der Bindung von Liganden des Carbonsäure-Typs nahe. Zur weiteren Interpretation werden in diesem Kapitel pKa-rechnungen zusammen mit ortsspezifischer Mutagenese und thermodynamischen Messungen eingesetzt. Diese lassen ein in der katalytischen Tasche befindliches Tyrosin (Tyr 48) für die Protonenaufnahme bei der Ligandbindung als wahrscheinlich erscheinen, da diese Seitenkette im NADP+-gebundenen Zustand zu bemerkenswertem Ausmaß deprotoniert vorliegt. In diesem Zusammenhang wird die Bindung von IDD 388, IDD 393, Tolrestat, Sorbinil und Fidarestat diskutiert.
In Kapitel 6 werden selektivitätsbestimmende Eigenschaften identifiziert, die die Bindung von Liganden an ALR2 energetisch günstiger erscheinen lassen als an die konkurrierende Isoform Aldehyd Reduktase (ALR1). Die zu diesem Zweck erstellten mutierten ALR2-Konstrukte wurden mit Hilfe von Kristallstrukturbestimmung und ITC auf ihre Einflussnahme bezüglich der Ligandenselektivität untersucht. Dazu diente ein Ligandensatz, der aus Zopolrestat, einem verwandten Uracil-Derivat, IDD 388, IDD 393, Sorbinil, Fidarestat und Tolrestat zusammengestellt wurde. Es konnte gezeigt werden, dass das Auftreten von Anpassungsvorgängen in der mutierten Bindetasche essentiell für die Einpassung der Liganden ist. Dieses lässt Rückschlüsse auf das Selektivitätsverhalten der Liganden zu. In vielen Fällen ist die Kristallstrukturbestimmung als schlussendlicher Beleg für die spezifische Wechselwirkung des Liganden mit dem Zielmolekül angesehen, da sie das genaue Wechselwirkungsmuster beider Bindungspartner repräsentiert. Diese weit verbreitete Vorgehensweise beruht auf der Annahme, dass eine Kristallstruktur eines gegebenen Protein-Ligand-Komplexes einzigartig und unabhängig von dem Protokoll ist, die zur Erzeugung der komplexierten Kristalle diente. In Kapitel 7 werden zwei Beispiele diskutiert, die zeigen, dass diese Annahmen nicht allgemein gültig sind.
2007-07-12
English
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