Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are the two enzymes of the polyol pathway. This pathway has been implicated in diabetic complications. Aldose reductase inhibitors (ARIs) have been shown to prevent or retard cataract, retinopathy, and neuropathy in animals. Clinical trials have been less successful and have pointed out the need to develop additional strategies of treatment. Our studies have focused on elucidating the structure and regulation of both enzymes of the polyol pathway. Using site-directed mutagenesis, we have now shown that some residues in the active site pocket of AR interact with ARIs. These data support the prediction that ARIs bind in the substrate binding site. Our data also indicate that the inhibitors have different orientations within this site because a differential affect was observed among the inhibitors. For example, tolrestat, a potent ARI, was virtually unaffected by mutant W111A but greatly affected by W20A. In contrast, ARI-509 was affected by W111A but not by W20A. These studies will be informative for structure-based drug design. Studies on SDH have included expression in the baculovirus system and purification of SDH for x-ray crystallography and analysis using SDH inhibitors. We have also investigated the gene structure and regulation of human AR and SDH. The basal activity of both promoters was established. In addition, we have found that the human AR promoter can be regulated by osmotic stress, and localization of the cis-elements responsible has yielded two sites that confer up to a 200-fold increase in luciferase reporter gene expression when transfected cells are exposed to hypertonic media. These studies will help define the mechanism by which AR is regulated at the gene level.
