Isolation of a Non-covalent Aldose Reductase–Nucleotide–Inhibitor Complex Katsumi Sugiyama, Zhou Chen, Yong S. Lee and Peter F. Kador* LABORATORY OF OCULAR THERAPEUTICS,NATIONAL EYE INSTITUTE,NATIONAL INSTITUTES OF HEALTH, BETHESDA, MD 20892-1850, U.S.A. ABSTRACT. 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'-imida- zolidine-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. BIOCHEM PHARMACOL 59;4:329 –336, 2000. © 2000 Elsevier Science Inc. KEY WORDS. aldose reductase; diabetes; aldose reductase inhibitors; mechanism of action; non-covalent binding Excess sorbitol accumulation catalyzed by aldose reductase (EC 1.1.1.21) has been linked to a number of ocular diabetic complications including keratopathy, cataract, and retinopathy [1, 2]. Administration of aldose reductase inhibitors at the onset of diabetes or galactosemia has been shown to prevent corneal epithelial and endothelial changes, osmotic cataract formation, and the selective destruction of retinal capillary pericytes and subsequent formation of microaneurysms and acellular vessels associ- ated with diabetic retinopathy [2, 3]. While a number of structurally diverse compounds inhibit aldose reductase significantly, many of these compounds possess poor phar- macokinetic properties or side-effects not associated with the specific inhibition of aldose reductase [4]. These side- effects range from hydantoin-related skin rash to induction of liver transaminases [5]. These problems reflect a need for the development of new, more potent inhibitors devoid of side-effects. The design of more potent and specific aldose reductase inhibitors requires an understanding of the interaction between aldose reductase bound to its nucleotide cofactor and its inhibitors and their mechanism of action. Insight into this has come from analyses of a series of crystallized human and porcine aldose reductase–NADP + –inhibitor complexes and molecular modeling studies [6 –14]. In ad- dition, specific non-covalent interactions between porcine aldose reductase complexed with NADP + and inhibitors have been investigated by electrospray mass spectroscopy [15–17]. Here, we report a method for the in vitro isolation of a non-covalent complex formed in solution by the interaction of human muscle or rat lens aldose reductase with either NADP + or NADPH and the aldose reductase inhibitors tolrestat, AL1576, or ponalrestat. MATERIALS AND METHODS Unless otherwise stated, all chemicals were of reagent grade, and HPLC solvents were of HPLC grade. NADP(H) was obtained from the Sigma Chemical Co. Microcon TM 10 filter units were obtained from Amicon, Inc. HMAR† was purchased from Wako BioProducts. RLAR expressed in Escherichia coli was purified as previously described [18]. The aldose reductase inhibitors AL1576 (2,7-difluorospiroflu- orene-9,5'-imidazolidine-2',4'-dione), sorbinil (S-6-fluoro- spirochroman-4,5'-imidazolidine-2',4'-dione), tolrestat {N- [(5-trifluoromethyl-6-methoxy-1-naphthalenyl)thioxom- ethyl]-N-methylglycine}, and ponalrestat {3-([4-bromo-2- fluorobenzyl]-4-oxo-3H-phthalazin-1-yl)acetic acid} were gifts from Alcon Laboratories, Pfizer Central Research, Wyeth-Ayerst Research, and ICI Americas, respectively. * Corresponding author: Peter F. Kador, Ph.D., Building 10, Room 10B-11, National Eye Institute, National Institutes of Health, 10 Center Dr., MSC 1850, Bethesda, MD 20892. Tel. (301) 496-6993; FAX (301) 402-2399. † Abbreviations: HMAR, recombinant human muscle aldose reductase; RLAR, recombinant rat lens aldose reductase; TFA, trifluoroacetic acid; and CHARMM, Chemistry at Harvard Macromolecular Mechanics. Received 26 March 1999; accepted 9 July 1999. Biochemical Pharmacology, Vol. 59, pp. 329 –336, 2000. ISSN 0006-2952/00/$–see front matter © 2000 Elsevier Science Inc. All rights reserved. PII S0006-2952(99)00332-9