Probing the inhibitor‐binding site of aldose reductase with site‐directed mutagenesis

Abstract

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, Ϫ79, Ϫ111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions.Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800Ϫ2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.Keywords : site-directed mutagenesis ; human aldose reductase; enzyme inhibition; polyol pathway ; diabetes.The polyol pathway has been implicated in the etiology of the secondary complications of diabetes. There are two enzymes in this pathway. The first, aldose reductase (AR), catalyzes the reduction of glucose to sorbitol and the second, sorbitol dehydrogenase, catalyzes the oxidation of sorbitol to fructose. AR has a relatively low affinity for glucose and during conditions of normal glycemia (80Ϫ110 mg/dl), where cellular glucose rapidly becomes phosphorylated via hexokinase, little substrate enters the pathway. During hyperglycemia, however, the cellular level of glucose greatly increases in tissues where glucose entry is independent of insulin. In these tissues which include the lens, retina, kidney and peripheral nerves, all sites of diabetes-induced

Aldose and aldehyde reductases: Correlation of molecular modeling and mass spectrometric studies on the binding of inhibitors to the active site

“…30,33 Replacing the hydrophobic residues Trp 22 and Trp 114 with Ala decreased the potential for nonbonded interactions. Except in the case of the binding of tolrestat to the Trp 114 to Ala mutant, the mutations resulted in 400-to 2,200-fold increases in IC 50 values, suggesting that hydrophobic interactions between the enzyme and inhibitor in solution contribute to the inhibitor's binding stability in the active site.…”

“…ALR1 activity was assayed spectrometrically in a Shimadzu UV-160A spectrophotometer by following the decrease in absorption of NADPH at 340 nm with dl-glyceraldehyde as substrate and according to published methodology. 30 For IC 50 measurements, the substrate concentration was maintained at 5 mM and a 1% DMSO was used with or without different concentrations of inhibitors. The temperature was maintained at 25°C and measurements were carried out in duplicates or triplicates with typical variations of less than 5%.…”

“…Zopolrestat was modeled in the active sites of porcine ALR1 and human ALR2 holoenzymes (PDB entries 1CWN and 1ADS), and tolrestat and sorbinil were modeled in the active site of human ALR2 by using a previously described procedure. 30 Briefly, each inhibitor molecule was manually docked in the active site of the holoenzyme in an orientation similar to that present in the crystal structure of the corresponding ternary complex of the human enzyme with zopolrestat 18 and of the eye-lens pig enzyme with tolrestat and sorbinil. 19 Water molecules in the active site of the holoenzyme coinciding with the inhibitor-binding position were removed.…”

Kinetic and Structural Characterization of the Glutathione-binding Site of Aldose Reductase

“…In most cases, the nature of inhibition by the glutathione analogs was uncompetitive versus the aldehyde substrate. Although for a simple, one-substrate/one-product reaction scheme, this would suggest that the inhibitors bind to the E⅐S complex, previous experiments have shown that several AR inhibitors such as sorbinil, tolrestat, and zopolrestat that do not display competitive inhibition (26,27) bind exclusively to the active site of the enzyme (27)(28)(29)(30). This is because the rate-limiting step in AR catalysis is the slow isomerization of the E⅐NADP binary complex (31).…”

Aldose reductase structures: implications for mechanism and inhibition

“…This pocket was shown to be closed in the ALR2-sorbinil complex but opened in the ALR2-tolrestat complex, which allowed tolrestat to bind within this pocket, albeit in a different conformation to the ALR2-zopolrestat structure [34]. The W111A mutant decreased the inhibition by 400-fold for zopolrestat but the W111Y mutant had no effect, which suggested that the effects of the Ala mutation were due to changes in the van der Waals interactions of the ALR2-ARI complex [37]. The most significant change in activity was observed for the W20A mutant, even though the number of eliminated van der Waals contacts was less than that for the W111A mutation.…”