Numerous animal studies indicate that aldose reductase inhibitors (ARIs) are beneficial for the prevention or amelioration of diabetic complications such as neuropathy, nephropathy and the ocular complications of cataract, retinopathy and keratopathy. To aid in the identification of novel potent ARIs, we have previously developed a screening method that is based on the formation of a non-covalent ternary tight-binding enzyme-inhibitor-nucleotide (AR-ARI-NADPH) complex that can be isolated using YM-10 filter units. Here, we report a modification of this method that permits us to rapidly identify tight binding ARIs that are isolated by denaturation from AR-ARI-NADPH complexes that are free of possible contamination resulting from the reaction of methanol with the YM-10 filter units. For the development of this procedure, nine structurally diverse ARIs were mixed with purified recombinant rat lens aldose reductase (RLAR) bound with NADPH to form tight-binding RLAR-ARI-NADPH complexes. These complexes were purified by high pressure Sephadex 75 size exclusion chromatography using ammonium acetate buffer and the formation of each complex was confirmed by electrospray ionisation mass spectrometry (ESI-MS). Each of the complexes was then denatured with methanol, rechromatographed on the size exclusion column, and the identity of the bound ARIs was confirmed by ESI-MS. The apparent ARI binding with aldose reductase to form a tight binding ARI complex appeared proportional to their IC 50 values. This procedure allows for the rapid identification of tight binding ARIs with apparent IC 50s<0.1μM.
- aldose reductase
- aldose reductase inhibitors
- diabetic complications
- electrospray ionisation mass spectrometry
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience