Towards Improved Aldose Reductase Inhibitors - Structural and Thermodynamic Investigation of Mutant and Wild Type Aldose Reductase Inhibitor Complexes
Rational drug design for flexible proteins like human aldose reductase comprises special challenges to find novel lead compounds or to improve known inhibitors. In this thesis, diverse aspects of the complexity of such a task were investigated to find promising new lead scaffolds and enhance the aff...
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|Zusammenfassung:||Rational drug design for flexible proteins like human aldose reductase comprises special challenges to find novel lead compounds or to improve known inhibitors. In this thesis, diverse aspects of the complexity of such a task were investigated to find promising new lead scaffolds and enhance the affinity of known aldose reductase inhibitors. Within different design cycles, the prediction of ligand binding modes in a binding pocket capable to adapt to a bound ligand is such a challenging task. A benzothiazole scaffold originating from a virtual screening run was kinetically and structurally evaluated. An unexpected binding mode in complex with wild-type aldose reductase and a new protein conformer provided the basis for a further drug design cycle to optimize the scaffold. The benzothiazole core was expanded to explicitly address an additional subpocket of the protein to enhance both affinity and selectivity over a closely related protein. Flexibility is a common feature of many proteins. For human aldose reductase, a variety of conformers are adopted with different inhibitors. In Chapter 3, the known inhibitors zopolrestat and IDD393 each in complex with a threonine 113 mutant and a benzothiazole inhibitor in complex with the wild type of aldose reductase were investigated with respect to the impact of inhibitor binding on protein conformation. Though the interaction to the mutated residue does not directly alter the binding mode of zopolrestat, a shift of its basic scaffold is induced and subsequently affects the interaction to a flexible loop and introduces disorder. With IDD393, two distinct binding site conformations resulting in different crystal forms can be directly compared. Improvements of the computational methods for affinity prediction from the structure of protein-ligand complexes requires a better understanding of the nature of molecular interactions and biomolecular recognition principles. In Chapter 4, the binding of two chemically closely related human aldose reductase inhibitors have been studied by high resolution X-ray analysis (0.92A-1.35A) and isothermal titration calorimetry against a series of single-site mutants of the wild-type protein. A crucial active site threonine, thought to be involved in a short bromine-to-oxygen halogen bond to the inhibitors in the wild type has been mutated to the structurally similar residues alanine, cysteine, serine, and valine. Overall, structurally the binding mode of the inhibitors is conserved; however, small but significant geometrical adaptations are observed as a consequence of the spatial and electronic changes at the mutation site. They involve the opening of a central bond angle and shifts in consequence of the lost or gained halogen bonds. Remarkably, the tiny structural changes are responded by partly strong modulation of the thermodynamic profiles. Even though the free energy of binding is maximally perturbed by only 7kJ/mol, much stronger modulations and shifts in the enthalpy and entropy signature are revealed which indicate a pronounced enthalpy/entropy compensation. However, facing these perturbances against the small structural changes, an explanatory correlation can be detected. This also provides deeper insights how single-site mutations can alter the selectivity profile of closely related ligands against a target protein. High resolution structural data of protein inhibitor complexes is the key to rational drug design. Synchrotron radiation allows for atomic resolution but is frequently accompanied by radiation damage to protein complexes. In Chapter 5, a human aldose reductase mutant complexed with a bromine substituted inhibitor was determined to atomic resolution. Though the radiation dose was moderate, a selective disruption of a bromine-inhibitor bond during the experiment was observed while the protein appears unaffected. A covalent bond to bromine is cleaved and the displaced atom is not scattered throughout the crystal but can most likely be assigned as a bromide ion to an additional difference electron density peak observed in the structure: The bromide relocates to an adjacent unoccupied site where promising interactions to protein residues stabilize its position. These findings were verified by a second similar structure determined with considerably higher radiation dose.|