Stereospecific interaction of a novel spirosuccinimide type aldose reductase inhibitor, AS-3201, with aldose reductase.
Aldose reductase (AR) is an NADPH-dependent enzyme implicated in diabetic complications. AS-3201 [(R)-(-)-2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3'-pyrrolidine-1,2',3,5'-tetrone] is a structurally novel and potent ARI with an inhibitor constant (K(i) = 10(-)(10) M) 2000-fold lower than that of its optical antipode (S-isomer). To elucidate the inhibition modes and the stereochemical differences in their inhibitory potencies, we examined the interaction of these R- and S-isomers with AR under physiological conditions. Enzyme kinetic analysis, which was performed by using physiological substrates at 37 degrees C, showed that both isomers selectively act on the E-NADP(+) complex in both the forward and reverse reactions of AR. However, fluorometric titration analysis demonstrated that the affinities of the isomers for the E-NADP(+) complex are about the same as those for the E-NADPH complex and the apoenzyme. These results suggested that the selective binding to the E-NADP(+) complex arises from the predominance of this enzyme form during steady-state turnover rather than from binding specificity. Both the competition with a known active site-directed ARI and the protective effect on AR inactivation by N-bromosuccinimide showed that the isomers bind to the active site of the enzyme, but the thermodynamic parameters for the binding to AR indicated that additional hydrogen bonds and/or van der Waals interactions contribute to the energetic stabilization in the E-R-isomer complex. Molecular modeling, together with the deductions from spectroscopic studies, suggested that the succinimide ring and the 4-bromo-2-fluorobenzyl group of the R-isomer are optimally located for formation of a hydrogen-bonding network with AR, and that the latter benzyl group is also effective for the differentiation between AR and aldehyde reductase (a closely related enzyme).
Kurono
M
M
Discovery Research Laboratories, Dainippon Pharmaceutical Company, Ltd., Enoki 33-94, Suita, Osaka 564-0053, Japan. masuo-kurono@dainippon-pharm.co.jp
Fujiwara
I
I
Yoshida
K
K
D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001974:Bromosuccinimide / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005461:Fluorine; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011719:Pyrazines / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013141:Spiro Compounds / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D017186:Titrimetry
D004791:Enzyme Inhibitors; D020035:Holoenzymes; D008024:Ligands; D011719:Pyrazines; D013141:Spiro Compounds; D013388:Succinimides; C032620:succinimide; D005461:Fluorine; D000449:Aldehyde Reductase; D001974:Bromosuccinimide; C115070:ranirestat