Isolation of a non-covalent aldose reductase-nucleotide-inhibitor complex.
A method for the isolation of an intact, non-covalent complex formed by the interaction of aldose reductase, NADP(H) nucleotide, and inhibitor has been developed to aid in the discovery and development of novel aldose reductase inhibitors. In the complexes isolated, both the carboxylic acid-containing inhibitor tolrestat and the spirohydantoin-containing inhibitor AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione) tightly bound in a 1:1 ratio to aldose reductase complexed with either NADPH or NADP+. Inhibitor binding to either the enzyme-NADP+ or enzyme-NADPH complex appeared to be equal and pH-dependent, with maximum binding observed at a pH range of 7 to 8.5 where the inhibitors are ionized. These results indicated that the charge state of the cofactor (NADPH vs NADP+) is not critical for inhibitor binding to aldose reductase. Molecular modeling studies suggested that His110 plays a crucial role in directing charged inhibitors containing either a carboxylate or an ionizable hydantoin group to the active site of aldose reductase by providing charge interaction.
Sugiyama
K
K
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-1850, USA.
Chen
Z
Z
Lee
Y S
YS
Kador
P F
PF
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005449:Fluorenes / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006827:Hydantoins / Q000378:metabolism / Q000494:pharmacology; D007908:Lens, Crystalline / Q000201:enzymology; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D009249:NADP / Q000302:isolation & purification / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism / Q000494:pharmacology; D010793:Phthalazines / Q000378:metabolism / Q000494:pharmacology; D051381:Rats
D004791:Enzyme Inhibitors; D005449:Fluorenes; D006827:Hydantoins; D009281:Naphthalenes; D010793:Phthalazines; C052241:imirestat; C040550:tolrestat; C042829:ponalrestat; D009249:NADP; D000449:Aldehyde Reductase