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 °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).