Studying Electrostatic Fields in Human Aldose Reductase with New Inhibitor as Probe

Abstract

Our research focuses on electrostatics in the system of human aldose reductase (hALR2), a 36 kDa aldo-keto reductase, which plays an important role in diabetes control. Vibrational Stark effect (VES) spectroscopy is utilized to measure the electrostatic fields near the active site of hALR2, using nitrile-containing inhibitors as the probe. Herein, a new hARL2 inhibitor was synthesized and bound to wild type hALR2 (wt_hALR2) with binding constant of 200 nM. Two vibrational absorption peaks were observed in the nitrile region when it bound to wt_hALR2, indicating the probe was experiencing two different environments. To explore the source of the two peaks, electrostatics calculations were performed based on crystal structures of hALR2 bound with similar inhibitors. The calculated projection of the protein electrostatic field also had a two-peak distribution. Analysis of trajectories suggests that they might be correlated with a possible hydrogen bond between the nitrile probe and a nearby residue threonine 113. To further test this assumption, IR spectra of the inhibitor bound to a series of mutants were taken; especially, the inhibitor bound to mutant hALR2_T113A had a single peak, which was also confirmed by simulations. This approach provides precise local information on electrostatic fields.