Identification and Characterization of Human Aldose Reductase Differential Inhibitors

The approach of aldose reductase (AKR1B1) differential inhibition is based on the possibility to inhibit the activity of the enzyme depending on the substrate undergoing transformation. This approach has been proposed to counteract damaging events linked to the activity of AKR1B1 in hyperglycemic conditions (i.e. polyols accumulation and generation of pro-inflammatory signals), leaving unaffected the ability of the enzyme to remove lipid peroxidation deriving toxic aldehydes. In this thesis two different approaches for the identification of AKR1B1 differential inhibitors from both natural and synthetic sources are reported. Zolfino beans water extracts were used as a starting material for the isolation, through hydrophobic interaction chromatography, of AKR1B1 differential inhibitors. The purified active fractions contained a diglycosylated kaempferol; the tentative identification, based on mass spectroscopy results, of this compound as kaempferol 3-O-β-D- glucopyranoside-(2→1)-O-β-D-xylopyranoside (leucoside) was not supported by the kinetic characterization of the inhibitory properties of commercial lecucoside. Thus, the attribution of the chemical structure to the differential inhibitor isolated from Zolfino beans remained at the moment an open question. The second approach deals with a High Throughput Screen (HTS) of a library of 1280 chemical compounds already known to exert different biological actions. An AKR1B1 assay to be performed in 384-well plates was thus optimized in order the HTS could be accomplished. The HTS allowed the selection of 4 promising compounds, that were characterized for their mechanisms of inhibition toward different AKR1B1 substrates, essentially confirming the differential inhibitory features, and tested for their ability to affect cultured human lens epithelial cells viability. Finally, two cellular models suitable to test the ability of molecules to impair AKR1B1 activity in cultured cells and the consequent NF-kB activation were set up.