title: Price for Opening the Transient Specificity Pocket in Human Aldose Reductase upon Ligand Binding: Structural, Thermodynamic, Kinetic, and Computational Analysis.
abstract: Insights into the thermodynamic and kinetic signature of the transient opening of a protein-binding pocket resulting from accommodation of suitable substituents attached to a given parent ligand scaffold are presented. As a target, we selected human aldose reductase, an enzyme involved in the development of late-stage diabetic complications. To recognize a large scope of substrate molecules, this reductase opens a transient specificity pocket. The pocket-opening step was studied by X-ray crystallography, microcalorimetry, and surface plasmon resonance using a narrow series of 2-carbamoyl-phenoxy-acetic acid derivatives. Molecular dynamics simulations suggest that pocket opening occurs only once an appropriate substituent is attached to the parent scaffold. Transient pocket opening of the uncomplexed protein is hardly recorded. Hydration-site analysis suggests that up to five water molecules entering the opened pocket cannot stabilize this state. Sole substitution with a benzyl group stabilizes the opened state, and the energetic barrier for opening is estimated to be ∼5 kJ/mol. Additional decoration of the pocket-opening benzyl substituent with a nitro group results in a huge enthalpy-driven potency increase; on the other hand, an isosteric carboxylic acid group reduces the potency 1000-fold, and binding occurs without pocket opening. We suggest a ligand induced-fit mechanism for the pocket-opening step, which, however, does not represent the rate-determining step in binding kinetics.
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, D-35032 Marburg, Germany.
authors.forename: Chris
authors.initials: C
authors.lastname: Rechlin
authors.affiliation: Institut für Pharmazeutische Chemie, Zentrum für Tumor- und Immunbiologie (ZTI), Philipps-Universität Marburg , Hans-Meerwein-Straße 3, 35043 Marburg, Germany.
authors.forename: Frithjof
authors.initials: F
authors.lastname: Scheer
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, D-35032 Marburg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Terwesten
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, D-35032 Marburg, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Wulsdorf
authors.affiliation: GE Healthcare Bio-Sciences AB , Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Ewa
authors.initials: E
authors.lastname: Pol
authors.affiliation: GE Healthcare Bio-Sciences AB , Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Veronica
authors.initials: V
authors.lastname: Fridh
authors.affiliation: Institut für Pharmazeutische Chemie, Zentrum für Tumor- und Immunbiologie (ZTI), Philipps-Universität Marburg , Hans-Meerwein-Straße 3, 35043 Marburg, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Toth
authors.affiliation: Institut für Pharmazeutische Chemie, Zentrum für Tumor- und Immunbiologie (ZTI), Philipps-Universität Marburg , Hans-Meerwein-Straße 3, 35043 Marburg, Germany.|Core Facility Medicinal Chemistry, Philipps-Universität Marburg , Hans-Meerwein-Straße 3, 35043 Marburg, Germany.
authors.forename: Wibke E
authors.identifier: 0000-0001-5671-6575
authors.initials: WE
authors.lastname: Diederich
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, D-35032 Marburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Heine
authors.affiliation: Institut für Pharmazeutische Chemie, Philipps-Universität Marburg , Marbacher Weg 6, D-35032 Marburg, Germany.
authors.forename: Gerhard
authors.identifier: 0000-0002-4913-390X
authors.initials: G
authors.lastname: Klebe
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry*; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D008024:Ligands; D000449:Aldehyde Reductase