AKR1B10: A potential target for cancer therapy

Aldo-keto reductase family 1 B10 (AKR1B10, also designated aldose reductase-like-1, ARL-1) is a novel protein identified from human hepatocellular carcinoma (HCC). This protein belongs to aldo-keto reductase superfamily, a group of proteins implicated in intracellular detoxification, cell carcinogenesis, and cancer therapeutics. AKR1B10 is primarily expressed in the colon and small intestine with low levels in the liver, thymus, prostate, and testis but overexpressed in the liver and lung cancer, making it a potential cancer diagnostic and/or prognostic marker. AKR1B10 could reduce retinals to retinols eliminating intracellular retinoic acid, a signaling molecule regulating cell proliferation and differentiation. AKR1B10 may impact the carcinogenesis process through controlling retinoic acid signaling.     

Potent and selective inhibition of the tumor marker AKR1B10 by bisdemethoxycurcumin: Probing the active site of the enzyme with molecular modeling and site-directed mutagenesis

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, shares high sequence identity with aldose reductase (AR), and was recently identified as a therapeutic target in the treatment of several types of cancer. We have compared the inhibitory effects of plant components on recombinant AKR1B10 and AR. AKR1B10 was inhibited by curcuminoids, magnolol, honokiol and resveratrol, with IC₅₀ values of 0.06-5 μM, which were lower than their values for AR. Among them, bisdemethoxycurcumin was the most potent competitive inhibitor (K_i = 22 nM) with the highest selectivity (85-fold versus AR), and acted as an effective inhibitor in cellular level. In contrast, demethoxycurcumin and curcumin showed >3-fold less potency and selectivity. Molecular docking studies of the curcuminoids in the AKR1B10-NADP⁺ complex and site-directed mutagenesis of the putative binding residues suggest that Gln114, Val301 and Gln303 are important for determining the inhibitory potency and selectivity of the curcuminoids.     

Chromene-3-carboxamide derivatives discovered from virtual screening as potent inhibitors of the tumour maker,AKR1B10

A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a therapeutic target in the treatment of several types of cancer. In order to identify potential leads for new inhibitors of AKR1B10, we adopted the virtual screening approach using the automated program icm, which resulted in the discovery of several chromene-3-carboxamide derivatives as potent competitive inhibitors. The most potent (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide inhibited the reductase activity of AKR1B10 with a K_i value of 2.7 nM, and the metabolism of farnesal and 4-hydroxynonenal in the AKR1B10-overexpressed cells from 0.1 μM with an IC₅₀ value equal to 0.8 μM.     

Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency

Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5β-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5β-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Δ⁴ double bond in 3-keto steroids and sterols to yield the 5β-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5β-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5β-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced K_m and V_max reminiscent of uncompetitive kinetics with 4-cholesten-7α-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5β-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.     

The hop-derived compounds xanthohumol,isoxanthohumol and 8-prenylnaringenin are tight-binding inhibitors of human aldo-keto reductases 1B1 and 1B10

Xanthohumol (XN), a prenylated chalcone unique to hops (Humulus lupulus) and two derived prenylflavanones, isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) gained increasing attention as potential anti-diabetic and cancer preventive compounds. Two enzymes of the aldo-keto reductase (AKR) superfamily are notable pharmacological targets in cancer therapy (AKR1B10) and in the treatment of diabetic complications (AKR1B1). Our results show that XN, IX and 8-PN are potent uncompetitive, tight-binding inhibitors of human aldose reductase AKR1B1 (K_i?=?15.08?μM, 0.34?μM, 0.71?μM) and of human AKR1B10 (K_i?=?20.11?μM, 2.25?μM, 1.95?μM). The activity of the related enzyme AKR1A1 was left unaffected by all three compounds. This is the first time these three substances have been tested on AKRs. The results of this study may provide a basis for further quantitative structure–activity relationship models and promising scaffolds for future anti-diabetic or carcinopreventive drugs.     

Activation of aldo-keto reductase family member 1B14 (AKR1B14) by bile acids: Activation mechanism and bile acid-binding site

Aldo-keto reductase (AKR) 1B14, a rat ortholog of mouse androgen-dependent vas deferens protein (AKR1B7), is involved in the synthesis of prostaglandin F_2α and detoxification of 4-oxononenal formed by lipid peroxidation. The NADPH-linked reductase activity of AKR1B14 was activated by various bile acids. Although the activation was increased by decreasing pH from 9.0 to 6.0, the concentrations giving maximum stimulation (2- to 18-fold) were 0.2-6.0 μM for bile acids at pH 7.4. Kinetic analyses of the activation by glycochenodeoxycholic acid in the forward and reverse reactions, together with fluorescence changes and protection against 4-oxononenal-induced inactivation by bile acid, indicate that the bile acid binds to the enzyme and its coenzyme binary complex as a non-essential activator. The bile acid binding to AKR1B14 mainly accelerates the NADP⁺ dissociation, the rate-limited step of the enzyme reaction. AKR1B7 was also activated by bile acids, but the activation was low and independent of pH. The mutagenesis of His269 and Leu267 of AKR1B14 into the corresponding residues (Arg and Pro, respectively) of AKR1B7 resulted in low and pH-independent activation by bile acids. The results, together with the docking of the bile acid in the recently determined crystal structure of AKR1B14, identify the bile acid-binding site of which His269 plays a key role in significant activation through its electrostatic interaction with the carboxyl group of bile acid, facilitating the release of NADP⁺.     

AKR1B10 is induced by hyperglycaemia and lipopolysaccharide in patients with diabetic nephropathy

Aldose reductase family member B10 (AKR1B10) belongs to the aldo–keto reductase gene superfamily and is closely related to aldose reductase (AKR1B1). It has been shown that AKR1B10 is present in many of the same human tissues as AKR1B1. The objective of this study was to investigate whether AKR1B10 has a role in diabetic nephropathy (DN) by investigating its response to high glucose and inflammation, both of which have been associated with the development and progression of DN. Expression levels of AKR1B10 were determined in peripheral blood mononuclear cells (PBMCs) obtained from 25 patients with type 1 diabetes and nephropathy, 25 without DN and 25 normal healthy controls that were exposed to high glucose (25 mM d-glucose) and also the inflammatory stressor lipopolysaccharide (LPS, 10 μm). Under high glucose and LPS conditions, there was a significant increase in the expression of AKR1B10 in the PBMCs from patients with DN compared to those without DN and the normal controls. In conclusion, these results suggest that AKR1B10 may have an important role in the development and progression of DN.     

Synthesis of non-prenyl analogues of baccharin as selective and potent inhibitors for aldo-keto reductase 1C3

Inhibitors of a human member (AKR1C3) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of prostatic and breast cancers. Baccharin [3-prenyl-4-(dihydrocinnamoyloxy)cinnamic acid], a component of propolis, was shown to be both potent (K_i 56 nM) and highly isoform-selective inhibitor of AKR1C3. In this study, a series of derivatives of baccharin were synthesized by replacing the 3-prenyl moiety with aryl and alkyl ether moieties, and their inhibitory activities for the enzyme were evaluated. Among them, two benzyl ether derivatives, 6m and 6n, showed an equivalent inhibitory potency to baccharin. The molecular docking of 6m in AKR1C3 has allowed the design and synthesis of (E)-3-{3-[(3-hydroxybenzyl)oxy]-4-[(3-phenylpropanoyl)oxy]phenyl}acrylic acid (14) with improved potency (K_i 6.4 nM) and selectivity comparable to baccharin. Additionally, 14 significantly decreased the cellular metabolism of androsterone and cytotoxic 4-oxo-2-nonenal by AKR1C3 at much lower concentrations than baccharin.     

The three-dimensional structure of AKR11B4, a glycerol dehydrogenase from Gluconobacter oxydans, reveals a tryptophan residue as an accelerator of reaction turnover

The NADP-dependent glycerol dehydrogenase (EC 1.1.1.72) from Gluconobacter oxydans is a member of family 11 of the aldo-keto reductase (AKR) enzyme superfamily; according to the systematic nomenclature within the AKR superfamily, the term AKR11B4 has been assigned to the enzyme. AKR11B4 is a biotechnologically attractive enzyme because of its broad substrate spectrum, combined with its distinctive regioselectivity and stereoselectivity. These features can be partially rationalized based on a 2-Å crystal structure of apo-AKR11B4, which we describe and interpret here against the functional complex structures of other members of family 11 of the AKR superfamily. The structure of AKR11B4 shows the AKR-typical (β/α)₈ TIM-barrel fold, with three loops and the C-terminal tail determining the particular enzymatic properties. In comparison to AKR11B1 (its closest AKR relative), AKR11B4 has a relatively broad binding cleft for the cosubstrate NADP/NADPH. In the crystalline environment, it is completely blocked by the C-terminal segment of a neighboring protomer. The structure reveals a conspicuous tryptophan residue (Trp23) that has to adopt an unconventional and strained side-chain conformation to permit cosubstrate binding. We predict and confirm by site-directed mutagenesis that Trp23 is an accelerator of (co)substrate turnover. Furthermore, we show that, simultaneously, this tryptophan residue is a critical determinant for substrate binding by the enzyme, while enantioselectivity is probably governed by a methionine residue within the C-terminal tail. We present structural reasons for these notions based on ternary complex models of AKR11B4, NADP, and either octanal, d-glyceraldehyde, or l-glyceraldehyde.     

A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli

Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from Anabaena sp. PCC7120 and adds on as 17^th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as— (i) conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii) excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde), and (iii) obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl) 4,-methyl phenol as the major products confirmed by GC-MS analysis.     

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His₆-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K_m and k_cat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min⁻¹, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

Downregulation of <Emphasis Type="Italic">AKR1B10</Emphasis> expression in colorectal cancer

Colorectal cancer is one of the most common cancers in the world. Changes in AKR1B1 and AKR1B10 expression levels, whose diagnostic value was previously shown for several other cancer types, were studied in colorectal tumors. These genes encode aldose reductases, members of the aldo-keto reductase superfamily, which comprises enzymes capable to reduce a range of aromatic and aliphatic aldehydes and ketones. They are also involved in retinoid metabolism and carcinogenesis. AKR1B1 and AKR1B10 mRNA levels were compared in paired specimens of normal and colorectal tumor tissues using RT-PCR and quantitative real-time PCR. For the first time, the downregulation of these genes was demonstrated in colorectal carcinoma. AKR1B10 expression was decreased in most tumor specimens (88%, 65/74) even at the early stages, and in more than 60% of cases mRNA levels were decreased more than 10-fold. AKR1B1 mRNA levels were decreased in 10% of specimens. Therefore, these two structurally similar genes show quite different mRNA expression patterns in colorectal cancer, suggestive of their different functional roles in the intestine. Significant downregulation of AKR1B10 expression can be considered a potential diagnostic marker of colorectal cancer.     

Kinetic studies of AKR1B10, human aldose reductase-like protein: Endogenous substrates and inhibition by steroids

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was identified as a biomarker of lung cancer, exhibiting high sequence identity with human aldose reductase (AKR1B1). Using recombinant AKR1B10 and AKR1B1, we compared their substrate specificity for biogenic compounds and inhibition by endogenous compounds and found the following unique features of AKR1B10. AKR1B10 efficiently reduced long-chain aliphatic aldehydes including farnesal and geranylgeranial, which are generated from degradation of prenylated proteins and metabolism of farnesol and geranylgeraniol derived from the mevalonate pathway. The enzyme oxidized aliphatic and aromatic alcohols including 20α-hydroxysteroids. In addition, AKR1B10 was inhibited by steroid hormones, bile acids and their metabolites, showing IC₅₀ values of 0.03-25 μM. Kinetic analyses of the alcohol oxidation and inhibition by the steroids and tolrestat, together with the docked model of AKR1B10-inhibitor complex, suggest that the inhibitory steroids and tolrestat bind to overlapping sites within the active site of the enzyme-coenzyme complex. Thus, we propose a novel role of AKR1B10 in controlling isoprenoid homeostasis that is important in cholesterol synthesis and cell proliferation through salvaging isoprenoid alcohols, as well as its metabolic regulation by endogenous steroids.     

Role of aldo‐keto reductase family 1 member B1 (AKR1B1) in the cancer process and its therapeutic potential

The role of aldo‐keto reductase family 1 member B1 (AKR1B1) in cancer is not totally clear but growing evidence is suggesting to have a great impact on cancer progression. AKR1B1 could participate in a complicated network of signalling pathways, proteins and miRNAs such as mir‐21 mediating mechanisms like inflammatory responses, cell cycle, epithelial to mesenchymal transition, cell survival and apoptosis. AKR1B1 has been shown to be mostly overexpressed in cancer. This overexpression has been associated with inflammatory mediators including nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB), cell cycle mediators such as cyclins and cyclin‐dependent kinases (CDKs), survival proteins and pathways like mammalian target of rapamycin (mTOR) and protein kinase B (PKB) or AKT, and other regulatory factors in response to reactive oxygen species (ROS) and prostaglandin synthesis. In addition, inhibition of AKR1B1 has been shown to mostly have anti‐cancer effects. Several studies have also suggested that AKR1B1 inhibition as an adjuvant therapy could render tumour cells more sensitive to anti‐cancer therapy or alleviate the adverse effects of therapy. AKR1B1 could also be considered as a potential cancer diagnostic biomarker since its promoter has shown high levels of methylation. Although pre‐clinical investigations on the role of AKR1B1 in cancer and the application of its inhibitors have shown promising results, the lack of clinical studies on AKR1B1 inhibitors has hampered the use of these drugs to treat cancer. Thus, there is a need to conduct more clinical studies on the application of AKR1B1 inhibitors as adjuvant therapy on different cancers.     

Synthesis and structure–activity relationship of 2-phenyliminochromene derivatives as inhibitors for aldo–keto reductase (AKR) 1B10

Inhibitors of a human member (AKR1B10) of the aldo–keto reductase superfamily are regarded as promising therapeutics for the treatment of cancer. Recently, we have discovered (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide (1) as the potent competitive inhibitor using the virtual screening approach, and proposed its 4-methoxy group on the 2-phenylimino moiety as an essential structural prerequisite for the inhibition. In this study, 18 derivatives of 1 were synthesized and their inhibitory potency against AKR1B10 evaluated. Among them, 7-hydroxy-2-(4-methoxyphenylimino)-2H-chromene-3-carboxylic acid benzylamide (5n) was the most potent inhibitor showing a K_i value of 1.3 nM. The structure–activity relationship of the derivatives indicated that the 7-hydroxyl group on the chromene ring, but not the 4-methoxy group, was absolutely required for inhibitory activity, The molecular docking of 5n in AKR1B10 and site-directed mutagenesis of the enzyme residues suggested that the hydrogen-bond interactions between the 7-hydroxyl group of 5n and the catalytic residues (Tyr49 and His111) of the enzyme, together with a π-stacking interaction of the benzylamide moiety of 5n with Trp220, are important for the potent inhibition.     

Murine aldo-keto reductase family 1 subfamily B: identification of AKR1B8 as an ortholog of human AKR1B10

Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.