title: Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase.
abstract: 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. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.
authors: Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
authors: Holger
authors: H
authors: Steuber
authors: Paul
authors: P
authors: Czodrowski
authors: Christoph A
authors: CA
authors: Sotriffer
authors: Gerhard
authors: G
authors: Klebe
mesh_terms: D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain; D048909:Diabetes Complications / Q000517:prevention & control; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D009249:NADP; D011485:Protein Binding; D011522:Protons; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D011522:Protons; D009249:NADP; D000449:Aldehyde Reductase