All in the family: aldose reductase and closely related aldo-keto reductases

Aldose reductase catalyzes the first step in the polyol pathway and is thought to be involved in the pathogenesis of diabetic complications. In addition to polyol synthesis, aldose reductase may have multiple other activities that intersect with signal processing and oxidative defense mechanisms. Multiple aldose reductase-like proteins have been discovered to have structures and catalytic properties that broadly overlap those of aldose reductase. This chapter will summarize new insights into properties and functions of aldose reductase and closely related members of the aldo-keto reductase enzyme superfamily.     

Aldose reductase structures: implications for mechanism and inhibition

During chronic hyperglycaemia, elevated vascular glucose level causes increased flux through the polyol pathway, which induces functional and morphological changes associated with secondary diabetic complications. Inhibitors of aldose reductase (ARIs) have been widely investigated as potential therapeutic agents, but to date only epalrestat is successfully marketed for treatment of diabetic neuropathy, in Japan. Promising compounds during in vitro studies or in trials with animal models have failed to proceed beyond clinical trials and to everyday use, due to a lack of efficacy or adverse side effects attributed to lack of inhibitor specificity and likely inhibition of the related aldehyde reductase (ALR1). Knowledge of the catalytic mechanism and structures of the current inhibitors complexed with ALR2 are means by which more specific and tightly bound inhibitors can be discovered. This review will provide an overview of the proposed catalytic mechanism and the current state of structure-based drug design.     

Strategies for the design of inhibitors of aldose reductase, an enzyme showing pronounced induced-fit adaptations

Aldose reductase is involved in the polyol pathway, catalyzing the reduction of glucose to sorbitol. However, due to pronounced binding site adaptations, the enzyme can operate on a broad palette of structurally diverse substrates ranging from small aliphatic and aromatic aldehydes up to steroid-type ligands. A comparative analysis of the presently accessible crystal structures of aldose reductase complexes reveals four binding-competent protein conformations. Additional relevant conformers are detected through molecular dynamics simulations. They indicate an equilibrium of several conformers which is shifted towards the binding-competent geometries upon ligand binding. Such a manifold system with several alternative binding site conformers requires some tailored concepts in virtual screening. We followed two strategies, both successfully suggesting new micromolar inhibitors. In a first attempt, we concentrated on one preferred conformer and performed a virtual screening, assuming that the binding pocket of aldose reductase adopts only this conformation. In a second approach, we followed a ligand superpositioning method. Ligands were extracted in their bound conformations from three different crystal structures, all accommodating the ligands with different active site conformations. After merging these ligands into one supermolecule, mutual alignments were computed, taking candidate ligands from a screening database. The latter strategy also retrieved several structurally new inhibitors of micromolar potency.     

Aldose metabolism in developing human fetal brain and liver

Summary Aldose reductase, sorbitol dehydrogenase, and glucose-6-phosphate dehydrogenase enzyme activities were studied in human foetal brain and liver at different periods of gestation. Aldose reductase activity in liver disappears after 16 weeks of gestation whereas sorbitol dehydrogenase keeps on increasing in liver as well as in brain. In utero, some glucose metabolism may be mediated through an active sorbitol pathway in human fetuses.Acknowledgments. These studies were supported by the Indian Council of Medical Research.     

Aldose metabolism in developing human fetal brain and liver

Aldose reductase, sorbitol dehydrogenase, and glucose-6-phosphate dehydrogenase enzyme activities were studied in human foetal brain and liver at different periods of gestation. Aldose reductase activity in liver disappears after 16 weeks of gestation whereas sorbitol dehydrogenase keeps on increasing in liver as well as in brain. In utero, some glucose metabolism may be mediated through an active sorbitol pathway in human fetuses.     

Inhibition of aldose reductase by dihydroflavonols inEngelhardtia chrysolepis and effects on other enzymes

Astilbin and neoastilbin, dihydroflavonol rhamnosides fromEngelhardtia chrysolepis, showed potent inhibition of lens aldose reductase. Kinetic analysis showed astilbin exhibited uncompetitive inhibition against bothdl-glyceraldehyde and NADPH. These taxifolin glycosides were selective inhibitors of aldose reductase with no inhibition of NADH oxidase.     

Potential aldose reductase inhibitors: 1,2,4-triazolidine-3,5-diones and 2-(3,4,5-trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidinedione

1,2,4-Triazolidine-3,5-diones and the 3,5-isoxazolidinedione were, observed to be, potent inhibitors of rat lens aldose reductase activity. In vivo in streptozotocin-diabetic rats, selected agents at 20 mg/kg/day, orally for 21 days reduced significantly the sorbitol levels of rbc, lens and sciatic nerves, suggesting that these derivatives may have some usefulness to treat clinical complications of diabetes mellitus.     

Diversity in enoyl-acyl carrier protein reductases

The enoyl-acyl carrier protein reductase (ENR) is the last enzyme in the fatty acid elongation cycle. Unlike most enzymes in this essential pathway, ENR displays an unusual diversity among organisms. The growing interest in ENRs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to specifically target their activity. The primary anti-tuberculosis drug, isoniazid, and the broadly used antibacterial compound, triclosan, both target this enzyme. In this review, we discuss the diversity of ENRs, and their inhibitors in the light of current research progress. Received 3 November 2008; received after revision 5 December 2008; accepted 8 December 2008     

Pancreatic islet β-cell deficit and glucose intolerance in rats with uninephrectomy

This study was performed to examine the effect of chronic renal impairment and renin-angiotensin system (RAS) activation induced by unilateral nephrectomy (UNX) on the development of pancreatic islet β-cell deficit and glucose intolerance. Sprague-Dawley rats were randomized into three groups: untreated UNX (n = 10), UNX treated with the angiotensin-converting enzyme inhibitor lisinopril (n = 8) and sham operation (n = 10). Blood glucose, serum insulin, renal function and histological changes of kidney and pancreas were examined 8 months postoperation. Compared with the sham rats, UNX rats developed renal impairment, insulin deficiency and glucose intolerance. Histological staining revealed an islet β-cell deficit associated with increased immunoreactivity for angiotensin and angiotensin type 1 receptor in UNX rats. Treatment with lisinopril significantly improved renal dysfunction, hyperglycemia, insulin secretion and islet RAS expression. These data suggest that chronic renal impairment and RAS activation may contribute to islet β-cell loss and glucose intolerance. RAS blockade may therefore prevent these disorders. Received 29 August 2007; received after revision 25 September 2007; accepted 27 September 2007     

Subatomic and atomic crystallographic studies of aldose reductase: implications for inhibitor binding

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 Å, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2¹), IDD 393 (resolution: 0.90 Å, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 Å, IC50: 9 nM, space group: P2¹) and minalrestat (resolution: 1.10 Å, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 0.3 Å). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl^- ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.     

Biochemical approaches to the study of plant-fungal interactions in arbuscular mycorrhiza

This communication compares some biochemical methods for quantifying colonization by arbuscular mycorrhizal (AM) fungi. The degree of mycorrhizal colonization can conveniently be measured by determining fungal specific sterols. AM-colonized plants show a specific synthesis of 24-methylene cholesterol and an enhanced level of campesterol (=24-methyl cholesterol). A gene probe for nitrate reductase, the key enzyme for nitrogen assimilation, has been developed, which allows the monitoring of the distribution of this enzyme in fungi. Among the phytohormones tested, only abscisic acid (ABA) is found at a considerably higher level in AM-colonized plants than in controls. The concentration of ABA is about twenty times higher in spores and hyphae of the AM fungusGlomus than in maize roots. Other phytohormones (auxins, cytokinins) do not show such alterations after mycorrhizal colonization. The roots of gramineous plants become yellow as a result of mycorrhizal colonization. The yellow pigment(s) formed is (are) deposited in larger quantities in the vacuole(s) of the root parenchyma and endodermis cells during the development of the gramineous plants. A substance isolated from such roots has now been identified as a C-14 carotenoid with two carboxylic groups, and named mycorradicin.     

Potential aldose reductase inhibitors: 1,2,4-triazolidine-3,5-diones and 2-(3,4,5-trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidinedione .

1,2,4-Triazolidine-3,5-diones and the 3,5-isoxazolidinedione were observed to be potent inhibitors of rat lens aldose reductase activity. In vivo in streptozotocin-diabetic rats, selected agents at 20 mg/kg/day, orally for 21 days reduced significantly the sorbitol levels of rbc, lens and sciatic nerves, suggesting that these derivatives may have some usefulness to treat clinical complications of diabetes mellitus.     

Type-2 copper-containing enzymes

Type-2 Cu sites are found in all the major branches of life and are often involved in the catalysis of oxygen species. Four type-2 Cu protein families are selected as model systems for review: amine oxidases, Cu monooxygenases, nitrite reductase/multicopper oxidase, and CuZn superoxide dismutase. For each model protein, the availability of multiple crystal structures and detailed enzymological studies provides a detailed molecular view of the type-2 Cu site and delineation of the mechanistic role of the Cu in biological function. Comparison of these model proteins leads to the identification of common properties of the Cu sites and insight into the evolution of the trinuclear active site found in multicopper oxidases. Received 6 July 2007; accepted 18 July 2007     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Summary Rat liver microsomal 3-hydroxy-3-methylgularyl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg⁺⁺. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg⁺⁺ by DPG were also observed with solubilized HMG-CoA reductase.This work was supported by Research Award # 697 G2-1 from the American Heart Association, Greater Los Angeles Affiliate, and by grant # 1R01 HL22672 from the National Institutes of Health. We thank M. Brun and M. Curtis for their excellent technical assistance.     

Plant thioredoxins: the multiplicity conundrum

Thioredoxins are small proteins distinguished by the presence of a conserved dicysteine active site. In oxidized thioredoxin, the two cysteines form a disulfide bond that is targeted by the enzyme thioredoxin reductase. Together with an electron donor, thioredoxin and thioredoxin reductase form the 'thioredoxin system' that is present in all organisms. Thioredoxins participate in dithiol/disulfide exchange reactions with a large range of cellular substrates. Higher plants possess a very complex thioredoxin profile consisting of at least two different thioredoxin systems that contain distinct, multigenic thioredoxin classes which have different intracellular localizations. In this review we summarise the current state of knowledge regarding the function of plant thioredoxins representing all systems and classes. Received 30 October 2001; received after revision 13 December 2001; accepted 17 December 2001     

The potential therapeutic role of statins in central nervous system autoimmune disorders

3-Hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins are widely used oral cholesterol-lowering drugs. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes conversion of HMG-CoA to L-mevalonate, a key intermediate in cholesterol synthesis. Certain metabolites of mevalonate are also involved in posttranslational modification of specific proteins involved in cell proliferation and differentiation. Thus, statins have important biologic effects that may be independent of their cholesterol-reducing properties. Recent studies indicate that statins have antiinflammatory and neuroprotective properties which may be beneficial in the treatment of multiple sclerosis as well as other central nervous system (CNS) neurodegenerative diseases. This article will outline current experimental evidence that may suggest potential clinical benefits for patients with CNS autoimmune disorders. Ultimately, clinical trials will have to determine the safety and efficacy of statins in this patient population.Received 17 April 2003; received after revision 21 May 2003; accepted 22 May 2003     

Human and yeast ζ-crystallins bind AU-rich elements in RNA

ζ-crystallins constitute a family of proteins with NADPH:quinone reductase activity found initially in mammalian lenses but now known to be present in many other organisms and tissues. Few proteins from this family have been characterized, and their function remains unclear. In the present work, ζ-crystallins from human and yeast (Zta1p) were expressed, purified and characterized. Both enzymes are able to reduce ortho-quinones in the presence of NADPH but are not active with 2-alkenals. Deletion of the ZTA1 gene makes yeast more sensitive to menadione and hydrogen peroxide, suggesting a role in the oxidative stress response. The human and yeast enzymes specifically bind to adenine-uracil rich elements (ARE) in RNA, indicating that both enzymes are ARE-binding proteins and that this property has been conserved in ζ-crystallins throughout evolution. This supports a role for ζ-crystallins as trans-acting factors that could regulate the turnover of certain mRNAs. Received 21 February 2007; received after revision 16 April 2007; accepted 23 April 2007 M. R. Fernández, S. Porté: These authors contributed equally to this work.     

Nitric oxide synthase reduces nitrite to NO under anoxia

Cultured bEND.3 endothelial cells show a marked increase in NO production when subjected to anoxia, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The rate of anoxic NO production exceeds basal unstimulated NO synthesis in normoxic cells. The anoxic release of NO is mediated by endothelial nitric oxide synthase (eNOS), can be abolished by inhibitors of NOS and is accompanied by consumption of intracellular nitrite. The anoxic NO release is unaffected by the xanthine oxidase inhibitor oxypurinol. The phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS, and its magnitude and duration suggests that the nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues. Received 20 August 2006; received after revision 21 September 2006; accepted 8 November 2006