Merging the binding sites of aldose and aldehyde reductase for detection of inhibitor selectivity-determining features

Inhibition of human aldose reductase (ALR2) evolved as a promising therapeutic concept to prevent late complications of diabetes. As well as appropriate affinity and bioavailability, putative inhibitors should possess a high level of selectivity for ALR2 over the related aldehyde reductase (ALR1). We investigated the selectivity-determining features by gradually mapping the residues deviating between the binding pockets of ALR1 and ALR2 into the ALR2 binding pocket. The resulting mutational constructs of ALR2 (eight point mutations and one double mutant) were probed for their influence towards ligand selectivity by X-ray structure analysis of the corresponding complexes and isothermal titration calorimetry (ITC). The binding properties of these mutants were evaluated using a ligand set of zopolrestat, a related uracil derivative, IDD388, IDD393, sorbinil, fidarestat and tolrestat. Our study revealed induced-fit adaptations within the mutated binding site as an essential prerequisite for ligand accommodation related to the selectivity discrimination of the ligands. However, our study also highlights the limits of the present understanding of protein-ligand interactions. Interestingly, binding site mutations not involved in any direct interaction to the ligands in various cases show significant effects towards their binding thermodynamics. Furthermore, our results suggest the binding site residues deviating between ALR1 and ALR2 influence ligand affinity in a complex interplay, presumably involving changes of dynamic properties and differences of the solvation/desolvation balance upon ligand binding.     

Probing flexibility and "induced-fit" phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations

Aldose reductase is a promising target for the treatment of diabetic complications, and as such, has become the focus of various drug design projects. As revealed by a survey of available crystal structures, the protein shows pronounced induced-fit effects upon ligand binding. Although helping to explain the enzyme's substrate promiscuity, phenomena of this kind are still responsible for significant complications in structure-based design efforts directed to aldose reductase. Accordingly, a deeper understanding of the principles governing conformational alterations in this enzyme would be of utmost practical importance. As a first step in addressing this issue, molecular dynamics (MD) simulations have been carried out. The ultrahigh resolution crystal structure of aldose reductase complexed with inhibitor IDD594 served as ideal starting point for a set of different simulations of nanosecond time scale: the native complexed state with bound inhibitor, the uncomplexed state (after removal of the inhibitor) at standard temperature, and the uncomplexed state at elevated temperature. The reference simulation of the complex exhibits extraordinary stability of the overall fold, whereas two distinct conformational substates are found for the binding-site region. In contrast, already at standard temperature pronounced changes are observed in the binding region during the simulation of the uncomplexed state. Leu300, for example, closes the access to the pocket opened by IDD594. On the other hand, conformations around the catalytic site are highly conserved, with the His110-Tyr48-NADP+ orientation being stabilized by a water molecule. Detailed analysis of the trajectories allows to reveal a set of distinct conformational substates that may prove useful as alternative structural templates in virtual screening for new aldose reductase inhibitors.     

Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.     

Virtual screening for inhibitors of human aldose reductase

The inhibition of aldose reductase (AR) provides an interesting strategy to prevent the complications of chronic diabetes. Although a large number of different AR inhibitors are known, very few of these compounds exhibit sufficient efficacy in clinical trials. We performed a virtual screening based on the ultrahigh resolution crystal structure of the inhibitor IDD594 in complex with human AR. AR operates on a large scale of structurally different substrates. To achieve this pronounced promiscuity, the enzyme can adapt rather flexibly to its substrates. Likewise, it has a similar adaptability for the binding of inhibitors. We applied a protocol of consecutive hierarchical filters to search the Available Chemicals Directory. In the first selection step, putative ligands were chosen that exhibit functional groups to anchor the anion-binding pocket of AR. Subsequently, a pharmacophore model based on the binding geometry of IDD594 and the mapping of the binding pocket in terms of putative "hot spots" of binding was applied as a second consecutive filter. In a third and final filtering step, the remaining candidate molecules were flexibly docked into the binding pocket of IDD594 with FlexX and ranked according to their estimated DrugScore values. Out of 206 compounds selected by this search and complemented by a cluster analysis and visual inspection, 9 compounds were selected and subjected to biological testing. Of these, 6 compounds showed IC50 values in the micromolar range. According to the proposed binding mode, the two inhibitors BTB02809 (IC50 = 2.4 +/- 0.5 microM) and JFD00882 (IC50 = 4.1 +/- 1.0 microM) both place a nitro group into the hydrophobic specificity pocket of human AR in an orientation coinciding with the position of the bromine atom of IDD594. The interaction of this Br with Thr113 has been identified as a key feature that is responsible for selectivity enhancement.     

Evidence for a novel binding site conformer of aldose reductase in ligand-bound state

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of how the slight change of a chemically extended side-chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships.     

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.     

The sorbinil trap: a predicted dead-end complex confirms the mechanism of aldose reductase inhibition

Kinetic and crystallographic studies have demonstrated that negatively charged aldose reductase inhibitors act primarily by binding to the enzyme complexed with oxidized nicotinamide dinucleotide phosphate (E.NADP(+)) to form a ternary dead-end complex that prevents turnover in the steady state. A recent fluorescence study [Nakano and Petrash (1996) Biochemistry 35, 11196-11202], however, has concluded that inhibition by sorbinil, a classic negatively charged aldose reductase inhibitor, results from binding to the enzyme complexed with reduced cofactor (E.NADPH) and not binding to E.NADP(+). To resolve this controversy, we present transient kinetic data which show unequivocally that sorbinil binds to E.NADP(+) to produce a dead-end complex, the so-called sorbinil trap, which prevents steady-state turnover in the presence of a saturating concentration of aldehyde substrate. The reported fluorescence binding results, which we have confirmed independently, are further shown to be fully consistent with the proposed sorbinil trap mechanism. Our conclusions are supported by KINSIM simulations of both pre-steady-state and steady-state reaction time courses in the presence and absence of sorbinil. Thus, while sorbinil binding indeed occurs to both E.NADPH and E.NADP(+), only the latter dead-end complex shows significant inhibition of the steady-state turnover rate. The effect of tight-binding kinetics on the inhibition patterns observed for zopolrestat, another negatively charged inhibitor, is further examined both experimentally and with KINSIM, with the conclusion that all reported aldose reductase inhibition can be rationalized in terms of binding of an alrestatin-like inhibitor at the active site, with no need to postulate a second inhibitor binding site.     

Carboxymethylation-induced activation of bovine lens aldose reductase.

Carboxymethylation of bovine lens aldose reductase with 10 mM iodoacetate for 1 h at 25 degrees C led to a more than 4-fold increase in kcat. Carboxymethylation led to a 3- to 5-fold increase in Km NADPH and Km D-glyceraldehyde, whereas Km L-glyceraldehyde increased approx. 30-fold. Activation of the enzyme on carboxymethylation was accompanied by a decrease in the sensitivity of the enzyme to inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), sorbinil (Kii increased from 0.4 to 109 microM) and NADP (Kis increased from 0.01 to 0.03 mM), but not tolrestat. Activation of the enzyme was almost completely prevented by NADPH and to a lesser extent by DL-glyceraldehyde. Carboxymethylation of the enzyme did not result in the generation of several partially oxidized enzyme species, indicating the absence of partially carboxymethylated forms. Primary deuterium isotope effects on the reduced enzyme were consistent with a preferred ordered kinetic reaction scheme, in which hydride transfer is not rate limiting. The hydride transfer step does not seem to be significantly affected by carboxymethylation, nor do changes in the substrate binding steps seem to contribute to the observed rate enhancement. Increase in the turnover number of the enzyme on carboxymethylation appears to be due to facilitation of the isomerization of the E:NADP binary complex. The differential effect of carboxymethylation on sorbinil and tolrestat suggests distinct inhibitor sites on the enzyme, an S-site that binds sorbinil and a T-site that binds tolrestat.     

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo-keto reductase (AKR) superfamily that is induced by the aldehyde-containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268: 5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 A resolution. Similar to aldose reductase and other AKRs, CHO reductase is an alpha/beta TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR-1 (mouse fibroblast growth factor-regulated protein), especially around the variable C-terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis.     

Ultrahigh resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 A

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.     

Aldehyde and aldose reductases from human placenta. Heterogeneous expression of multiple enzyme forms

Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).     

Prevention of neural myoinositol depletion in diabetic rats by aldose reductase inhibition with tolrestat

The effect of the aldose reductase inhibitor tolrestat on the sugar and polyol contents in the sciatic nerve was investigated in male Wistar and Sprague-Dawley rats rendered diabetic with streptozocin. At a daily oral dose of 5 mg/kg, given for 10 days before and for 14 days after streptozocin injection, tolrestat completely prevented the accumulation of sorbitol and the depletion of myoinositol.     

Inhibition of human brain aldose reductase and hexonate dehydrogenase by alrestatin and sorbinil

Abstract: Human brain aldose reductase and hexonate dehydrogenase are inhibited by alrestatin (AY 22,284) and sorbinil (CP 45,634). Inhibition by alrestatin is noncompetitive for both enzymes, and slightly stronger for hexonate dehydrogenase ( K _I values 52-250 μ M ) than for aldose reductase ( K _I values 170-320 μ M ). Sorbinil inhibits hexonate dehydrogenase far more potently than aldose reductase, K _I values being 5 μ M for hexonate dehydrogenase and 150 μ M for aldose reductase. The inhibition of hexonate dehydrogenase by sorbinil is noncompetitive with respect to both aldehyde and NADPH substrates, and is thus kinetically similar to the inhibition by alrestatin. However, sorbinil inhibition of aldose reductase is uncompetitive with respect to glyceraldehyde and noncompetitive with NADPH as the varied substrate. Inhibition of human brain aldose reductase by these two inhibitors is much less potent than that reported for the enzyme from other sources.     

Purified rat lens aldose reductase. Polyol production in vitro and its inhibition by aldose reductase inhibitors.

The production of polyols in vitro by highly purified aldose reductase (EC 1.1.1.21) was monitored by g.l.c. In the presence of NADPH aldose reductase reduced glucose, galactose and xylose to the respective polyols sorbitol, galactitol and xylitol. The rates of formation of these polyols closely mirrored the Km values for the substrates obtained from kinetic measurements that monitored the rate of disappearance of NADPH. No polyol production occurred in the absence of purified aldose of purified aldose reductase, and analysis by g.l.c. revealed only the presence of unchanged monosaccharides. Addition of the aldose reductase inhibitor sorbinil to purified rat lens aldose reductase incubated with xylose in the presence of NADPH resulted in decreased xylitol production. However, aldose reductase inhibitors produced no effect in altering the rate of Nitro Blue Tetrazolium formation from either glucose or xylose, indicating that the observed inhibition in vitro does not result from a free-radical-scavenger effect.     

Spirohydantoin derivatives of thiopyrano[2,3-b]pyridin-4(4H)-one as potent in vitro and in vivo aldose reductase inhibitors

The 2,3-dihydrospiro[4H-thiopyrano[2,3-b]pyridin-4,4'-imidazolidine]-2',5'-dione 3 and its 7-methyl analogue 4 were synthesized and tested for their ability to inhibit aldose reductase (ALR2). To expand the structure-activity relationships, the sulfone 5 and the acetic acid derivative 7 were also prepared and tested. Compounds 3 and 4 proved to be potent ALR2 inhibitors, with IC50 values in the submicromolar range (0.96 and 0.94 microM, respectively) similar to that of sorbinil (0.65 microM). Moreover, compound 3 was found to be highly potent in preventing cataract development in severely galactosemic rats, like tolrestat, when administered as an eyedrop solution. Docking simulations of both R- and S-isomers of 3 into the ALR2 crystal structure were carried out to guide, prospectively, the design of new analogues.     

Expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying soaking and co-crystallisation conditions

In structure-based drug design, accurate crystal structure determination of protein-ligand complexes is of utmost importance in order to elucidate the binding characteristics of a putative lead to a given target. It is the starting point for further design hypotheses to predict novel leads with improved properties. Often, crystal structure determination is regarded as ultimate proof for ligand binding providing detailed insight into the specific binding mode of the ligand to the protein. This widely accepted practise relies on the assumption that the crystal structure of a given protein-ligand complex is unique and independent of the protocol applied to produce the crystals. We present two examples indicating that this assumption is not generally given, even though the composition of the mother liquid for crystallisation was kept unchanged: Multiple crystal structure determinations of aldose reductase complexes obtained under varying crystallisation protocols concerning soaking and crystallisation exposure times were performed resulting in a total of 17 complete data sets and ten refined crystal structures, eight in complex with zopolrestat and two complexed with tolrestat. In the first example, a flip of a peptide bond is observed, obviously depending on the crystallisation protocol with respect to soaking and co-crystallisation conditions. This peptide flip is accompanied by a rupture of an H-bond formed to the bound ligand zopolrestat. The indicated enhanced local mobility of the complex is in agreement with the results of molecular dynamics simulations. As a second example, the aldose reductase-tolrestat complex is studied. Unexpectedly, two structures could be obtained: one with one, and a second with four inhibitor molecules bound to the protein. They are located in and near the binding pocket facilitated by crystal packing effects. Accommodation of the four ligand molecules is accompanied by pronounced shifts concerning two helices interacting with the additional ligands.     

An Engineered Disulfide Bond Reversibly Traps the IgE-Fc3–4 in a Closed,Nonreceptor Binding Conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcϵRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcϵRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcϵRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.     

Structural and thermodynamic studies of simple aldose reductase-inhibitor complexes

The competitive inhibition constants of series of inhibitors related to phenylacetic acid against both wild-type and the doubly mutanted C298A/W219Y aldose reductase have been measured. Van't Hoff analysis shows that these acids bind with an enthalpy near -6.8 kcal/mol derived from the electrostatic interactions, while the 100-fold differences in binding affinity appear to be largely due to entropic factors that result from differences in conformational freedom in the unbound state. These temperature studies also point out the difference between substrate and inhibitor binding. X-ray crystallographic analysis of a few of these inhibitor complexes both confirms the importance of a previously described anion binding site and reveals the hydrophobic nature of the primary binding site and its general plasticity. Based on these results, N-glycylthiosuccinimides were synthesized to demonstrate their potential in studies that probe distal binding sites. Reduced alpha-lipoic acid, an anti-oxidant and therapeutic for diabetic complications, was shown to bind aldose reductase with a binding constant of 1 microM.     

Effect of Sorbinil and Ascorbic Acid on myo-Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose

Abstract: The effect of long-term (2 weeks) exposure to 0–50 m M glucose and 0–1 m M sorbitol on myo -inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo -inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo -Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 m M glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 m M sorbinil. Ascorbic acid also prevented the reduction in myo -inositol uptake due to excess extracellular glucose, at 3 and 30 µ M concentrations, but not at 300 µ M . Neither sorbinil nor ascorbic acid could prevent the alterations in myo -inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo -inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo -inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.     

Role of nonenzymatic glycosylation in experimental cataract formation

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [³H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.