Price for Opening the Transient Specificity Pocket in Human Aldose Reductase upon Ligand Binding Structural, Thermodynamic, Kinetic, and Computational Analysis

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

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA Modifying Enzyme TGT

Fragment based lead discovery was applied to tRNA guanine transglycosylase, an enzyme modifying post transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella s virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1 recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1 recognition pocket. We synthesized 6 carboxamido , 6 hydrazido , and 4 guanidino benzimidazoles to target the opened pocket, including a dihydro imidazoquinazoline with a propyn 1 yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGT