Which Properties Allow Ligands to Open and Bind to the Transient Binding Pocket of Human Aldose Reductase?

Abstract

The transient specificity pocket of aldose reductase only opens in response to specific ligands. This pocket may offer an advantage for the development of novel, more selective ligands for proteins with similar topology that lack such an adaptive pocket. Our aim was to elucidate which properties allow an inhibitor to bind in the specificity pocket. A series of inhibitors that share the same parent scaffold but differ in their attached aromatic substituents were screened using ITC and X-ray crystallography for their ability to occupy the pocket. Additionally, we investigated the electrostatic potentials and charge distribution across the attached terminal aromatic groups with respect to their potential to bind to the transient pocket of the enzyme using ESP calculations. These methods allowed us to confirm the previously established hypothesis that an electron-deficient aromatic group is an important prerequisite for opening and occupying the specificity pocket. We also demonstrated from our crystal structures that a pH shift between 5 and 8 does not affect the binding position of the ligand in the specificity pocket. This allows for a comparison between thermodynamic and crystallographic data collected at different pH values.

In Search of Differential Inhibitors of Aldose Reductase

“…The search for new inhibitors of AKR1B1 is an active and constantly evolving field of research and every year several new molecules for the inhibition of AKR1B1 are designed and proposed [11,[47][48][49][50].…”

“…This region changes its own conformation depending on whether or not the NADPH cofactor is present and it can assume an "open" conformation, in the absence of NADPH or a "closed" conformation in the presence of the cofactor [5]. In the active site of the enzyme two regions can be identified: a rigid region, the so-called called "anion-binding pocket" containing residues Trp-20, Val-47, Tyr-48, His-110, Trp-111 and a more flexible hydrophobic region, called "specificity pocket", containing residues Thr-113, Phe-115, Phe-122, Cys-303 and Tyr-309 [6][7][8][9][10][11].…”

Dissecting the Activity of Catechins as Incomplete Aldose Reductase Differential Inhibitors through Kinetic and Computational Approaches

“…Thus, despite displaying different stability, different EI complexes may be generated starting from different enzyme conformations, which are in turn affected by different substrates. These data acknowledged the high adaptability of AKR1B1 in interacting with quite different molecules, acting both as substrates and as inhibitors [ 9 , 10 , 11 , 12 , 41 , 42 , 43 ]. It is, however, hard to predict whether the lower stability of the EI complex built from an “open specificity pocket” conformation of the enzyme ( Figure 9 B) with respect to the binary complex coming from a “closed specificity pocket” conformation ( Figure 9 A) is sufficient to rule out its generation, thus giving a rationale to the uncompetitive action of GCG and CG towards HNE and GSHNE reduction.…”

Identifying protein conformational states in the Protein Data Bank: Toward unlocking the potential of integrative dynamics studies

“…Many structures of this protein have been independently solved with a variety of ligands, providing information on the conformational heterogeneity within the holo state. 83 Individual PDB entries fail to capture the structural heterogeneity in the β-sheet region spanning Val121-Arg156, but our pipeline can separate the only non-liganded structure in the PDB (1XGD) from all other ligand-bound chains. Superposition of all chains highlights a structural deviation in the unliganded structure within the Pro211-Asp230 loop.…”