Aldo-keto reductase 1B10 as a Carcinogenic but Not a Prognostic Factor in Colorectal Cancer.

Colorectal cancer (CRC) is the leading cause of cancer death, but little is known about its etiopathology. Aldo-keto reductase 1B10 (AKR1B10) protein is primarily expressed in intestinal epithelial cells, but lost in colorectal cancer tissues. This study revealed that AKR1B10 may not be a prognostic but an etiological factor in colorectal tumorigenesis. Using a tissue microarray, we investigated the expression of AKR1B10 in tumor tissues of 592 colorectal cancer patients with a mean follow-up of 25 years. Results exhibited that AKR1B10 protein was undetectable in 374 (63.13%), weakly positive in 146 (24.66%), and positive 72 (12.16%) of 592 tumor tissues. Kaplan-Meier analysis showed that AKR1B10 expression was not correlated with overall survival or disease-free survival. Similar results were obtained in various survival analyses stratified by clinicopathological parameters. AKR1B10 was not correlated with tumor T-pathology, N-pathology, TNM stages, cell differentiation and lymph node/regional/distant metastasis either. However, AKR1B10 silencing in culture cells enhanced carbonyl induced protein and DNA damage; and in ulcerative colitis tissues, AKR1B10 deficiency was associated acrolein-protein lesions. Together this study suggests that AKR1B10 downregulation may not be a prognostic but a carcinogenic factor of colorectal cancer.

Structural and biochemical evidence that ATP inhibits the cancer biomarker human aldehyde dehydrogenase 1A3.

Human aldehyde dehydrogenase (ALDH) participates in the oxidative stress response and retinoid metabolism, being involved in several diseases, including cancer, diabetes and obesity. The ALDH1A3 isoform has recently elicited wide interest because of its potential use as a cancer stem cell biomarker and drug target. We report high-resolution three-dimensional ALDH1A3 structures for the apo-enzyme, the NAD+ complex and a binary complex with ATP. Each subunit of the ALDH1A3-ATP complex contains one ATP molecule bound to the adenosine-binding pocket of the cofactor-binding site. The ATP complex also shows a molecule, putatively identified as a polyethylene glycol aldehyde, covalently bound to the active-site cysteine. This mimics the thioacyl-enzyme catalytic intermediate, which is trapped in a dead enzyme lacking an active cofactor. At physiological concentrations, ATP inhibits the dehydrogenase activity of ALDH1A3 and other isoforms, with a Ki value of 0.48 mM for ALDH1A3, showing a mixed inhibition type against NAD+. ATP also inhibits esterase activity in a concentration-dependent manner. The current ALDH1A3 structures at higher resolution will facilitate the rational design of potent and selective inhibitors. ATP binding to ALDH1A3 enables activity modulation by the energy status of the cell and metabolic reprogramming, which may be relevant in several disease conditions.

Expansion of the 4-(Diethylamino)benzaldehyde Scaffold to Explore the Impact on Aldehyde Dehydrogenase Activity and Antiproliferative Activity in Prostate Cancer.

Aldehyde dehydrogenases (ALDHs) are overexpressed in various tumor types including prostate cancer and considered a potential target for therapeutic intervention. 4-(Diethylamino)benzaldehyde (DEAB) has been extensively reported as a pan-inhibitor of ALDH isoforms, and here, we report on the synthesis, ALDH isoform selectivity, and cellular potencies in prostate cancer cells of 40 DEAB analogues; three analogues (14, 15, and 16) showed potent inhibitory activity against ALDH1A3, and two analogues (18 and 19) showed potent inhibitory activity against ALDH3A1. Significantly, 16 analogues displayed increased cytotoxicity (IC50 = 10-200 µM) compared with DEAB (>200 µM) against three different prostate cancer cell lines. Analogues 14 and 18 were more potent than DEAB against patient-derived primary prostate tumor epithelial cells, as single agents or in combination treatment with docetaxel. In conclusion, our study supports the use of DEAB as an ALDH inhibitor but also reveals closely related analogues with increased selectivity and potency.

Perspective on the Structural Basis for Human Aldo-Keto Reductase 1B10 Inhibition.

Human aldo-keto reductase 1B10 (AKR1B10) is overexpressed in many cancer types and is involved in chemoresistance. This makes AKR1B10 to be an interesting drug target and thus many enzyme inhibitors have been investigated. High-resolution crystallographic structures of AKR1B10 with various reversible inhibitors were deeply analyzed and compared to those of analogous complexes with aldose reductase (AR). In both enzymes, the active site included an anion-binding pocket and, in some cases, inhibitor binding caused the opening of a transient specificity pocket. Different structural conformers were revealed upon inhibitor binding, emphasizing the importance of the highly variable loops, which participate in the transient opening of additional binding subpockets. Two key differences between AKR1B10 and AR were observed regarding the role of external loops in inhibitor binding. The first corresponded to the alternative conformation of Trp112 (Trp111 in AR). The second difference dealt with loop A mobility, which defined a larger and more loosely packed subpocket in AKR1B10. From this analysis, the general features that a selective AKR1B10 inhibitor should comply with are the following: an anchoring moiety to the anion-binding pocket, keeping Trp112 in its native conformation (AKR1B10-like), and not opening the specificity pocket in AR.

Design, Synthesis, Biological Evaluation and In Silico Study of Benzyloxybenzaldehyde Derivatives as Selective ALDH1A3 Inhibitors.

Aldehyde dehydrogenase 1A3 (ALDH1A3) has recently gained attention from researchers in the cancer field. Several studies have reported ALDH1A3 overexpression in different cancer types, which has been found to correlate with poor treatment recovery. Therefore, finding selective inhibitors against ALDH1A3 could result in new treatment options for cancer treatment. In this study, ALDH1A3-selective candidates were designed based on the physiological substrate resemblance, synthesized and investigated for ALDH1A1, ALDH1A3 and ALDH3A1 selectivity and cytotoxicity using ALDH-positive A549 and ALDH-negative H1299 cells. Two compounds (ABMM-15 and ABMM-16), with a benzyloxybenzaldehyde scaffold, were found to be the most potent and selective inhibitors for ALDH1A3, with IC50 values of 0.23 and 1.29 µM, respectively. The results also show no significant cytotoxicity for ABMM-15 and ABMM-16 on either cell line. However, a few other candidates (ABMM-6, ABMM-24, ABMM-32) showed considerable cytotoxicity on H1299 cells, when compared to A549 cells, with IC50 values of 14.0, 13.7 and 13.0 µM, respectively. The computational study supported the experimental results and suggested a good binding for ABMM-15 and ABMM-16 to the ALDH1A3 isoform. From the obtained results, it can be concluded that benzyloxybenzaldehyde might be considered a promising scaffold for further drug discovery aimed at exploiting ALDH1A3 for therapeutic intervention.

Structural and kinetic features of aldehyde dehydrogenase 1A (ALDH1A) subfamily members, cancer stem cell markers active in retinoic acid biosynthesis.

Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.

Engineering aldo-keto reductase 1B10 to mimic the distinct 1B15 topology and specificity towards inhibitors and substrates, including retinoids and steroids.

The aldo-keto reductase (AKR) superfamily comprises NAD(P)H-dependent enzymes that catalyze the reduction of a variety of carbonyl compounds. AKRs are classified in families and subfamilies. Humans exhibit three members of the AKR1B subfamily: AKR1B1 (aldose reductase, participates in diabetes complications), AKR1B10 (overexpressed in several cancer types), and the recently described AKR1B15. AKR1B10 and AKR1B15 share 92% sequence identity, as well as the capability of being active towards retinaldehyde. However, AKR1B10 and AKR1B15 exhibit strong differences in substrate specificity and inhibitor selectivity. Remarkably, their substrate-binding sites are the most divergent parts between them. Out of 27 residue substitutions, six are changes to Phe residues in AKR1B15. To investigate the participation of these structural changes, especially the Phe substitutions, in the functional features of each enzyme, we prepared two AKR1B10 mutants. The AKR1B10 m mutant carries a segment of six AKR1B15 residues (299-304, including three Phe residues) in the respective AKR1B10 region. An additional substitution (Val48Phe) was incorporated in the second mutant, AKR1B10mF48. This resulted in structures with smaller and more hydrophobic binding pockets, more similar to that of AKR1B15. In general, the AKR1B10 mutants mirrored well the specific functional features of AKR1B15, i.e., the different preferences towards the retinaldehyde isomers, the much higher activity with steroids and ketones, and the unique behavior with inhibitors. It can be concluded that the Phe residues of loop C (299-304) contouring the substrate-binding site, in addition to Phe at position 48, strongly contribute to a narrower and more hydrophobic site in AKR1B15, which would account for its functional uniqueness. In addition, we have investigated the AKR1B10 and AKR1B15 activity toward steroids. While AKR1B10 only exhibits residual activity, AKR1B15 is an efficient 17-ketosteroid reductase. Finally, the functional role of AKR1B15 in steroid and retinaldehyde metabolism is discussed.

Inhibitors of aldehyde dehydrogenases of the 1A subfamily as putative anticancer agents: Kinetic characterization and effect on human cancer cells.

Aldehyde dehydrogenases (ALDHs) are enzymes catalyzing the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. High ALDH activity has been related to some important features of cancer stem cells. ALDH1A enzymes, involved in the retinoic acid signaling pathway, are promising drug targets for cancer therapy, and the design of selective ALDH1A inhibitors has a growing pharmacological interest. In the present work, two already known compounds (DEAB and WIN 18,446) and novel thiazolidinedione and pyrimido quinoline acetic acid derivatives (compounds 5a and 64, formerly described as aldo-keto reductase inhibitors) were tested as inhibitors of the ALDH1A enzymes (namely, ALDH1A1, ALDH1A2 and ALDH1A3) as a first step to develop some potential drugs for cancer therapy. The inhibitory capacity of these compounds against the ALDH1A activity was characterized in vitro by using purified recombinant proteins. The IC50 values of each compound were determined indicating that the most potent inhibitors against ALDH1A1, ALDH1A2 and ALDH1A3 were DEAB, WIN 18,446 and compound 64, respectively. Type of inhibition and Ki values were determined for DEAB against ALDH1A1 (competitive, Ki = 0.13 µM) and compound 64 against ALDH1A3 (non-competitive, Ki = 1.77 µM). The effect of these inhibitors on A549 human lung cancer cell viability was assessed, being compound 64 the only inhibitor showing an important reduction of cell survival. We also tested the effect of the ALDH substrate, retinaldehyde, which was cytotoxic above 10 µM. This toxicity was enhanced in the presence of DEAB. Both DEAB and compound 64 were able to inhibit the ALDH1A activity in A549 cells. The current work suggests that, by blocking ALDH activity, drug inactivation may be avoided. Thus these results may be relevant to design novel combination therapies to fight cancer cell chemoresistance, using both enzyme inhibitors and chemotherapeutic agents.

Design, synthesis, structure-activity relationships and X-ray structural studies of novel 1-oxopyrimido[4,5-c]quinoline-2-acetic acid derivatives as selective and potent inhibitors of human aldose reductase.

Human aldose reductase (AKR1B1, AR) is a key enzyme of the polyol pathway, catalyzing the reduction of glucose to sorbitol at high glucose concentrations, as those found in diabetic condition. Indeed, AKR1B1 overexpression is related to diabetes secondary complications and, in some cases, with cancer. For many years, research has been focused on finding new AKR1B1 inhibitors (ARIs) to overcome these diseases. Despite the efforts, most of the new drug candidates failed because of their poor pharmacokinetic properties and/or unacceptable side effects. Here we report the synthesis of a series of 1-oxopyrimido[4,5-c]quinoline-2-acetic acid derivatives as novel ARIs. IC50 assays and X-ray crystallographic studies proved that these compounds are promising hits for further drug development, with high potency and selectivity against AKR1B1. Based on the determined X-ray structures with hit-to-lead compounds, we designed and synthesized a second series that yielded lead compound 68 (Kiappvs. AKR1B1 = 73 nM). These compounds are related to the previously reported 2-aminopyrimido[4,5-c]quinolin-1(2H)-ones, which exhibit antimitotic activity. Regardless of their similarity, the 2-amino compounds are unable to inhibit AKR1B1 while the 2-acetic acid derivatives are not cytotoxic against fibrosarcoma HT-1080 cells. Thus, the replacement of the amino group by an acetic acid moiety changes their biological activity, improving their potency as ARIs.

Structural basis for the inhibition of AKR1B10 by the C3 brominated TTNPB derivative UVI2008.

UVI2008, a retinoic acid receptor (RAR) ß/γ agonist originated from C3 bromine addition to the parent RAR pan-agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), is also a selective inhibitor of aldo-keto reductase family member 1B10 (AKR1B10). Thus, it might become a lead drug for the design of compounds targeting both activities, as an AKR1B10 inhibitor and RAR agonist, which could constitute a novel therapeutic approach against cancer and skin-related diseases. Herein, the X-ray structure of the methylated Lys125Arg/Val301Leu AKR1B10 (i.e. AKME2MU) holoenzyme in complex with UVI2008 was determined at 1.5 Å resolution, providing an explanation for UVI2008 selectivity against AKR1B10 (IC50 = 6.1 µM) over the closely related aldose reductase (AR, IC50 = 70 µM). The carboxylic acid group of UVI2008 is located in the anion-binding pocket, at hydrogen-bond distance of catalytically important residues Tyr49 and His111. The inhibitor bromine atom can only fit in the wider active site of AKR1B10, mainly because of the native Trp112 side-chain orientation, not possible in AR. In AKR1B10, Trp112 native conformation, and thus UVI2008 binding, is facilitated through interaction with Gln114. IC50 analysis of the corresponding Thr113Gln mutant in AR confirmed this hypothesis. The elucidation of the binding mode of UVI2008 to AKR1B10, along with the previous studies on the retinoid specificity of AKR1B10 and on the stilbene retinoid scaffold conforming UVI2008, could indeed be used to foster the drug design of bifunctional antiproliferative compounds.

IDD388 Polyhalogenated Derivatives as Probes for an Improved Structure-Based Selectivity of AKR1B10 Inhibitors.

Human enzyme aldo-keto reductase family member 1B10 (AKR1B10) has evolved as a tumor marker and promising antineoplastic target. It shares high structural similarity with the diabetes target enzyme aldose reductase (AR). Starting from the potent AR inhibitor IDD388, we have synthesized a series of derivatives bearing the same halophenoxyacetic acid moiety with an increasing number of bromine (Br) atoms on its aryl moiety. Next, by means of IC50 measurements, X-ray crystallography, WaterMap analysis, and advanced binding free energy calculations with a quantum-mechanical (QM) approach, we have studied their structure-activity relationship (SAR) against both enzymes. The introduction of Br substituents decreases AR inhibition potency but improves it in the case of AKR1B10. Indeed, the Br atoms in ortho position may impede these drugs to fit into the AR prototypical specificity pocket. For AKR1B10, the smaller aryl moieties of MK181 and IDD388 can bind into the external loop A subpocket. Instead, the bulkier MK184, MK319, and MK204 open an inner specificity pocket in AKR1B10 characterized by a π-π stacking interaction of their aryl moieties and Trp112 side chain in the native conformation (not possible in AR). Among the three compounds, only MK204 can make a strong halogen bond with the protein (-4.4 kcal/mol, using QM calculations), while presenting the lowest desolvation cost among all the series, translated into the most selective and inhibitory potency AKR1B10 (IC50 = 80 nM). Overall, SAR of these IDD388 polyhalogenated derivatives have unveiled several distinctive AKR1B10 features (shape, flexibility, hydration) that can be exploited to design novel types of AKR1B10 selective drugs.

Structural Determinants of the Selectivity of 3-Benzyluracil-1-acetic Acids toward Human Enzymes Aldose Reductase and AKR1B10.

The human enzymes aldose reductase (AR) and AKR1B10 have been thoroughly explored in terms of their roles in diabetes, inflammatory disorders, and cancer. In this study we identified two new lead compounds, 2-(3-(4-chloro-3-nitrobenzyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (JF0048, 3) and 2-(2,4-dioxo-3-(2,3,4,5-tetrabromo-6-methoxybenzyl)-3,4-dihydropyrimidin-1(2H)-yl)acetic acid (JF0049, 4), which selectively target these enzymes. Although 3 and 4 share the 3-benzyluracil-1-acetic acid scaffold, they have different substituents in their aryl moieties. Inhibition studies along with thermodynamic and structural characterizations of both enzymes revealed that the chloronitrobenzyl moiety of compound 3 can open the AR specificity pocket but not that of the AKR1B10 cognate. In contrast, the larger atoms at the ortho and/or meta positions of compound 4 prevent the AR specificity pocket from opening due to steric hindrance and provide a tighter fit to the AKR1B10 inhibitor binding pocket, probably enhanced by the displacement of a disordered water molecule trapped in a hydrophobic subpocket, creating an enthalpic signature. Furthermore, this selectivity also occurs in the cell, which enables the development of a more efficient drug design strategy: compound 3 prevents sorbitol accumulation in human retinal ARPE-19 cells, whereas 4 stops proliferation in human lung cancer NCI-H460 cells.

Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase.

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

Structural analysis of sulindac as an inhibitor of aldose reductase and AKR1B10.

Aldose reductase (AR, AKR1B1) and AKR1B10 are enzymes implicated in important pathologies (diabetes and cancer) and therefore they have been proposed as suitable targets for drug development. Sulindac is the metabolic precursor of the potent non-steroidal anti-inflammatory drug (NSAID) sulindac sulfide, which suppresses prostaglandin production by inhibition of cyclooxygenases (COX). In addition, sulindac has been found to be one of the NSAIDs with higher antitumoral activity, presumably through COX inhibition. However, sulindac anticancer activity could be partially mediated through COX-independent mechanisms, including the participation of AR and AKR1B10. Previously, it had been shown that sulindac and sulindac sulfone were good AR inhibitors and the structure of the ternary complex with NADP(+) and sulindac was described (PDB ID 3U2C). In this work, we determined the three-dimensional structure of AKR1B10 with sulindac and established structure-activity relationships (SAR) of sulindac and their derivatives with AR and AKR1B10. The difference in the IC50 values for sulindac between AR (0.36 µM) and AKR1B10 (2.7 µM) might be explained by the different positioning and stacking interaction given by Phe122/Phe123, and by the presence of two buried and ordered water molecules in AKR1B10 but not in AR. Moreover, SAR analysis shows that the substitution of the sulfinyl group is structurally allowed in sulindac derivatives. Hence, sulindac and its derivatives emerge as lead compounds for the design of more potent and selective AR and AKR1B10 inhibitors.

Identification of a novel polyfluorinated compound as a lead to inhibit the human enzymes aldose reductase and AKR1B10: structure determination of both ternary complexes and implications for drug design.

Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved (α/ß)8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-NADP(+)-JF0064 complex has been determined at 0.85 Šresolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallographic structure of the corresponding AKR1B10-NADP(+)-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts.

A missense mutation in ALDH1A3 causes isolated microphthalmia/anophthalmia in nine individuals from an inbred Muslim kindred.

Nine affected individuals with isolated anophthalmia/microphthalmia from a large Muslim-inbred kindred were investigated. Assuming autosomal-recessive mode of inheritance, whole-genome linkage analysis, on DNA samples from four affected individuals, was undertaken. Homozygosity mapping techniques were employed and a 1.5-Mbp region, homozygous in all affected individuals, was delineated. The region contained nine genes, one of which, aldehyde dehydrogenase 1 (ALDH1A3), was a clear candidate. This gene seems to encode a key enzyme in the formation of a retinoic-acid gradient along the dorsoventral axis during an early eye development and the development of the olfactory system. Sanger sequence analysis revealed a missense mutation, causing a substitution of valine (Val) to methionine (Met) at position 71. Analyzing the p.Val71Met missense mutation using standard open access software (MutationTaster online, PolyPhen, SIFT/PROVEAN) predicts this variant to be damaging. Enzymatic activity, studied in vitro, showed no changes between the mutated and the wild-type ALDH1A3 protein.

Biocatalytic production of alpha-hydroxy ketones and vicinal diols by yeast and human aldo-keto reductases.

The α-hydroxy ketones are used as building blocks for compounds of pharmaceutical interest (such as antidepressants, HIV-protease inhibitors and antitumorals). They can be obtained by the action of enzymes or whole cells on selected substrates, such as diketones. We have studied the enantiospecificities of several fungal (AKR3C1, AKR5F and AKR5G) and human (AKR1B1 and AKR1B10) aldo-keto reductases in the production of α-hydroxy ketones and diols from vicinal diketones. The reactions have been carried out with pure enzymes and with an NADPH-regenerating system consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase. To ascertain the regio and stereoselectivity of the reduction reactions catalyzed by the AKRs, we have separated and characterized the reaction products by means of a gas chromatograph equipped with a chiral column and coupled to a mass spectrometer as a detector. According to the regioselectivity and stereoselectivity, the AKRs studied can be divided in two groups: one of them showed preference for the reduction of the proximal keto group, resulting in the S-enantiomer of the corresponding α-hydroxy ketones. The other group favored the reduction of the distal keto group and yielded the corresponding R-enantiomer. Three of the AKRs used (AKR1B1, AKR1B10 and AKR3C1) could produce 2,3-butanediol from acetoin. We have explored the structure/function relationships in the reactivity between several yeast and human AKRs and various diketones and acetoin. In addition, we have demonstrated the utility of these AKRs in the synthesis of selected α-hydroxy ketones and diols.

X-ray structure of the V301L aldo-keto reductase 1B10 complexed with NADP(+) and the potent aldose reductase inhibitor fidarestat: implications for inhibitor binding and selectivity.

Only one crystal structure is currently available for tumor marker AKR1B10, complexed with NADP(+) and tolrestat, which is an aldose reductase inhibitor (ARI) of the carboxylic acid type. Here, the X-ray structure of the complex of the V301L substituted AKR1B10 holoenzyme with fidarestat, an ARI of the cyclic imide type, was obtained at 1.60Å resolution by replacement soaking of crystals containing tolrestat. Previously, fidarestat was found to be safe in phase III trials for diabetic neuropathy and, consistent with its low in vivo side effects, was highly selective for aldose reductase (AR or AKR1B1) versus aldehyde reductase (AKR1A1). Now, inhibition studies showed that fidarestat was indeed 1300-fold more selective for AR as compared to AKR1B10, while the change of Val to Leu (found in AR) caused a 20-fold decrease in the IC50 value with fidarestat. Structural analysis of the V301L AKR1B10-fidarestat complex displayed enzyme-inhibitor interactions similar to those of the AR-fidarestat complex. However, a close inspection of both the new crystal structure and a computer model of the wild-type AKR1B10 complex with fidarestat revealed subtle changes that could affect fidarestat binding. In the crystal structure, a significant motion of loop A was observed between AR and V301L AKR1B10, linked to a Phe-122/Phe-123 side chain displacement. This was due to the presence of the more voluminous Gln-303 side chain (Ser-302 in AR) and of a water molecule buried in a subpocket located at the base of flexible loop A. In the wild-type AKR1B10 model, a short contact was predicted between the Val-301 side chain and fidarestat, but would not be present in AR or in V301L AKR1B10. Overall, these changes could contribute to the difference in inhibitory potency of fidarestat between AR and AKR1B10.

Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARß/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket.

Biological role of aldo-keto reductases in retinoic Acid biosynthesis and signaling.

Several aldo-keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low K(m) and k(cat) values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in ß-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.