David Robert Brittain

(2,6-dimethylphenylsulphonyl)nitromethane: A new structural type of aldose reductase inhibitor which follows biphasic kinetics and uses an allosteric binding site

Many of the complications of diabetes seem to be due to aldose reductase (aldehyde reductase 2, ALR2) catalysing the increased conversion of glucose to sorbitol. Therapy with aldose reductase inhibitors (ARIs) could, therefore, decrease the development of diabetic complications. (2,6-Dimethylphenylsulphonyl)nitromethane (ICI 215918) is an example from a newly discovered class of ARIs, and we here describe its kinetic properties. Preparations of bovine lens ALR2 exhibit biphasic kinetics with respect to glucose and various inhibitors including ICI 215918. The inhibitor sensitive form (ALR2S) has a higher affinity for glucose than does the inhibitor insensitive form (ALR2I). Only ALR2S was characterized in detail because ALR2I activity is very low at physiological levels of glucose and is difficult to measure with accuracy. Aldehyde reductase (ALR1) is the most closely related enzyme to ALR2. Inhibition of ALR1 was, therefore, investigated in order to assess the specificity of ICI 215918. The values of Ki and Kies (dissociation constants for inhibitor from enzyme-inhibitor and enzyme-inhibitor-substrate complexes, respectively) for ICI 215918 with bovine kidney ALR1 and bovine lens ALR2S have been determined. When glucose is varied, the compound is an uncompetitive inhibitor of ALR2S (Kies = 0.10 microM and Ki is much greater than Kies), indicating that ICI 215918 associates with an allosteric site on the enzyme. These kinetic characteristics would cause a decrease in the concentration required to give 50% inhibition when glucose levels rise during hyperglycaemia. ICI 215918 is a mixed noncompetitive inhibitor of ALR1 (Ki = 10 microM and Kies = 1.8 microM) when glucuronate is varied. Thus, the compound has up to 100-fold specificity in favour of ALR2S relative to ALR1. Therapeutic interest has now centred upon at least three distinct structural types of ARIs: spirohydantoins, acetic acids and sulphonylnitromethanes. Using one representative of each type, we have demonstrated kinetic competition for inhibition of ALR2S. This observation strongly suggests that the different inhibitors use overlapping binding sites.

Inhibition of Aldose Reductase By (2, 6-Dimethylphenylsulphonyl)Nitromethane: Possible Implications for the Nature of an Inhibitor Binding Site and A Cause of Biphasic Kinetics

Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.