Involvement of porcine and human carbonyl reductases in the metabolism of epiandrosterone, 11-oxygenated steroids, neurosteroids, and corticosteroids.

Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C18/C19-steroids,11-keto- and 11ß-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) Km values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/ß-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3ß-HSD and 3α-HSD, respectively). Additionally, 17ß-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/ß-dihydro-C21-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5ß-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/ß-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/ß-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower Km values (0.3-2.9 µM) for the 3-keto-C21-steroids than pCBR-N1 (Km=10-36 µM). The reduced products of the 3-keto-C21-steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C18/C19/C21-steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/ß,17ß-HSDs.

Mining and tailor-made engineering of a novel keto reductase for asymmetric synthesis of structurally hindered γ- and δ-lactones.

A novel carbonyl reductase from Hyphopichia burtoni (HbKR) was discovered by gene mining. HbKR is a NADPH-dependent dual function enzyme with reduction and oxidation activity belonging to SDR superfamily. HbKR strictly follows Prelog priority in the reduction of long-chain aliphatic keto acids/esters containing remote carbonyl groups, such as 4-oxodecanoic acid and 5-oxodecanoic acid, producing (S)-γ-decalactone and (S)-δ-decalactone in >99 % e.e. Tailor-made engineering of HbKR was conducted to improve its catalytic efficiency. Variant F207A/F86M was obtained with specific activity of 8.37 U/mg toward 5-oxodecanoic acid, which was 9.7-fold of its parent. Employing F207A/F86M, 100 mM 5-oxodecanoic acid could be reduced into optically pure (S)-δ-decalactone. Molecular docking analysis indicates that substitution of aromatic Phe with smaller residues renders sufficient space for accommodating substrates in a more stable conformation. This study offers an efficient biocatalyst for the biosynthesis of (S)-lactones, and provides guidance for engineering carbonyl reductases toward structurally hindered substrates.

Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Efficient biosynthesis of Vibegron intermediate using a novel carbonyl reductase based on molecular modification of hydrogen bonding network regulation.

Vibegron is a novel, potent, highly selective ß3-adrenergic receptor agonist for the treatment of overactive bladder with higher therapeutic capacity and lower side effects. Methyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropanoate ((2S,3R)-aminohydroxy ester) is a key chiral intermediate for the synthesis of Vibegron. A novel carbonyl reductase from Exiguobacterium sp. s126 (EaSDR6) was isolated using data mining technology from GenBank database with preferable catalytic activity. Hydrogen bond network regulation was performed using site-directed saturation mutagenesis and combination mutagenesis. The mutant EaSDR6A138L/S193A was obtained with the activity improvement by 4.58 folds compared with the wild type EaSDR6. The Km of EaSDR6A138L/S193A was decreased from 1.57 mM to 0.67 mM, kcat was increased by 2.17 folds, and the overall catalytic efficiency kcat/Km was increased by 5.07 folds. The organic-aqueous biphasic bioreaction system for the asymmetric synthesis of (2S,3R)-aminohydroxy ester was constructed for the first time. Under the substrate concentration of 150 g/L, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99%, and the spatiotemporal yield was 1.55 g/(L·h·g DCW) after 12 h reaction. While the substrate concentration was increased to 200 g/L and the reaction lasted for 36 h, the yield of (2S,3R)-aminohydroxy ester was > 99.99%, the e.e. was > 99.99% and the spatiotemporal yield was 1.05 g/(L·h·g DCW). The substrate concentration and spatiotemporal yield were higher than ever reported.

Synthesis, activity, metabolic stability and cell permeability of new cytosolic phospholipase A2α inhibitors with 1-indolyl-3-phenoxypropan-2-one structure.

Cytosolic phospholipase A2α (cPLA2α), the key enzyme of the arachidonic acid cascade, is considered to be an interesting target for the development of new anti-inflammatory drugs. Potent inhibitors of the enzyme include indole-5-carboxylic acids with propan-2-one residues in position 1 of the indole. Previously, it was found that central pharmacophoric elements of these compounds are their ketone and carboxylic acid groups, which unfortunately are subject to pronounced metabolism by carbonyl reductases and glucuronosyltransferases, respectively. Here we show that the metabolic stability of these inhibitors can be improved by introducing alkyl substituents in the vicinity of the ketone group or by increasing their rigidity. Furthermore, permeability tests with Caco-2 cells revealed that the indole derivatives have only low permeability, which can be attributed to their affinity to efflux transporters. Among other things, the polar ketone group in the center of the molecules seems to be a decisive factor for their reverse transport. After its removal, the permeability increased significantly. The enhancement in metabolic stability and permeability achieved by the structural variations carried out was accompanied by a more or less pronounced decrease in the inhibitory potency of the compounds against cPLA2α.

Peripheralized sepiapterin reductase inhibition as a safe analgesic therapy.

The development of novel analgesics for chronic pain in the last 2 decades has proven virtually intractable, typically failing due to lack of efficacy and dose-limiting side effects. Identified through unbiased gene expression profiling experiments in rats and confirmed by human genome-wide association studies, the role of excessive tetrahydrobiopterin (BH4) in chronic pain has been validated by numerous clinical and preclinical studies. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthases, and alkylglycerol monooxygenase so a lack of BH4 leads to a range of symptoms in the periphery and central nervous system (CNS). An ideal therapeutic goal therefore would be to block excessive BH4 production, while preventing potential BH4 rundown. In this review, we make the case that sepiapterin reductase (SPR) inhibition restricted to the periphery (i.e., excluded from the spinal cord and brain), is an efficacious and safe target to alleviate chronic pain. First, we describe how different cell types that engage in BH4 overproduction and contribute to pain hypersensitivity, are themselves restricted to peripheral tissues and show their blockade is sufficient to alleviate pain. We discuss the likely safety profile of peripherally restricted SPR inhibition based on human genetic data, the biochemical alternate routes of BH4 production in various tissues and species, and the potential pitfalls to predictive translation when using rodents. Finally, we propose and discuss possible formulation and molecular strategies to achieve peripherally restricted, potent SPR inhibition to treat not only chronic pain but other conditions where excessive BH4 has been demonstrated to be pathological.

Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system.

Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3 , coevolved thermostability, and activity. Compared with LsCRM3 , the catalytic efficiency kcat /KM of LsCRM3 -E145A (LsCRM4 ) was increased from 6.6 to 21.9 s-1 mM-1 . Moreover, E145A prolonged the half-life t1/2 at 40°C from 4.1 to 117 h, T m ${T}_{m}$ was increased by 5°C, T 50 30 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% eeP , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT , the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

Construction of Bi-Enzyme Self-Assembly Clusters Based on SpyCatcher/SpyTag for the Efficient Biosynthesis of (R)-Ethyl 2-hydroxy-4-phenylbutyrate.

Cascade reactions catalyzed by multi-enzyme systems are important in science and industry and can be used to synthesize drugs and nutrients. In this study, two types of macromolecules of bi-enzyme self-assembly clusters (BESCs) consisting of carbonyl reductase (CpCR) and glucose dehydrogenase (GDH) were examined. Stereoselective CpCR and GDH were successfully fused with SpyCatcher and SpyTag, respectively, to obtain four enzyme modules, namely: SpyCatcher-CpCR, SpyCatcher-GDH, SpyTag-CpCR, and SpyTag-GDH, which were covalently coupled in vitro to form two types of hydrogel-like BESCs: CpCR-SpyCatcher-SpyTag-GDH and GDH-SpyCatcher-SpyTag-CpCR. CpCR-SpyCatcher-SpyTag-GDH showed a better activity and efficiently converted ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl(R)2-hydroxy-4-phenylbutanoate ((R)-HPBE), while regenerating NADPH. At 30 °C and pH 7, the conversion rate of OPBE with CpCR-SpyCatcher-SpyTag-GDH as a catalyst reached 99.9%, with the ee% of (R)-HPBE reaching above 99.9%. This conversion rate was 2.4 times higher than that obtained with the free bi-enzyme. The pH tolerance and temperature stability of the BESCs were also improved compared with those of the free enzymes. In conclusion, bi-enzyme assemblies were docked using SpyCatcher/SpyTag to produce BESCs with a special structure and excellent catalytic activity, improving the catalytic efficiency of the enzyme.

Bergapten attenuates microglia-mediated neuroinflammation and ischemic brain injury by targeting Kv1.3 and Carbonyl reductase 1.

Microglia-mediated neuroinflammation plays a vital role in the pathogenesis of ischemic stroke, which serves as a prime target for developing novel therapeutic agent. However, feasible and effective agents for controlling neuroinflammation are scarce. Bergapten were acknowledged to hold therapeutic potential in restricting inflammation in multiple diseases, including peripheral neuropathy, migraine headaches and osteoarthritis. Here, we aimed to investigate the impact of bergapten on microglia-mediated neuroinflammation and its therapeutic potential in ischemic stroke. Our study demonstrated that bergapten significantly reduced the expression of pro-inflammatory cytokines and the activation of NF-κB signaling pathway in LPS-stimulated primary microglia. Mechanistically, bergapten suppressed cellular potassium ion efflux by inhibiting Kv1.3 channel and inhibits the degradation of Carbonyl reductase 1 induced by LPS, which might contribute to the anti-inflammatory effect of bergapten. Furthermore, bergapten suppressed microglial activation and post-stroke neuroinflammation in an experimental stroke model, leading to reduced infarct size and improved functional recovery. Thus, our study identified that bergapten might be a potential therapeutic compound for the treatment of ischemic stroke.

Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol.

Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase LsCR for the synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a "small and smart" mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant LsCRM3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) LsCR, the catalytic activity of LsCRM3 was increased by 4.7 times, the catalytic efficiency kcat/KM value was increased by 2.9 times, and the half-life t1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% (v/v) isopropanol, LsCRM3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e.) in 11 h with a space-time yield (STY) as high as 809 g/L∙d.

The crystal structure of the S154Y mutant carbonyl reductase from Leifsonia xyli explains enhanced activity for 3,5-bis(trifluoromethyl)acetophenone reduction.

Carbonyl reductase from Leifsonia xyli (LXCAR, UniProtKB - T2FLN4) can stereoselectively catalyze the reduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) to its corresponding alcohol, (R)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((R)-BTPE), which is a valuable chiral intermediate for the synthesis of antiemetic drugs, Aprepitant and Fosaprepitant. Moreover, this protein was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters. Even though molecular modelling of this protein was done by Wang et al. (2014), a crystal structure has not yet obtained. In this study, a single mutant, S154Y, which was shown to have higher catalytic activity toward BTAP than that of the wild type, was overexpressed in Escherichia coli BL21 (DE3), purified, and crystallized. The crystal structure of LXCAR-S154Y explains how the mutant enzyme can work with BTAP more efficiently than wild type carbonyl reductase. Furthermore, the structure explains why LXCAR-S154Y can use either NADH or NADPH efficiently as a cofactor, as well as elucidates a proton relay system present in the enzyme.

Identification and functional analysis of carbonyl reductases related to tetrahydrobiopterin synthesis in the silkworm, Bombyx mori.

The superfamily of short-chain dehydrogenases/reductases (SDRs) is crucial in biosynthetic and signalling pathways, in which the carbonyl reductases (CBRs) subfamily is important in the biosynthesis of tetrahydrobiopterin (BH4). BH4 is an essential coenzyme for animals, and its deficiency can lead to neurological diseases. There are few reports on CBRs involved in BH4 synthesis of silkworms, Bombyx mori. Here, we identified 67 SDR genes in B. mori (BmSDR) through whole genome survey for the first time. Based on bioinformatics analyses and KEGG verification, four BmCBRs that may be related to BH4 synthesis were further characterized and functionally analysed. The results showed these four genes were high expressed in the head and gonads of ah09 (a lem mutant with defective BH4 synthesis). Enzyme activity, BH4 content and the related gene expression levels after intracellular interference with BmCBR and the main catalytic enzymes sepiapterin reductase of B. mori (BmSpr) in the de novo pathway of BH4 showed BmCBR2 plays a role in the salvage pathway. BmCBR3 and BmCBR4 regulate BH4 synthesis through the alternative pathway. Among the four pathways of silkworm BH4 synthesis, the de novo pathway occupies the dominant position, followed by the alternative pathway and salvage pathway. According to the overexpression of BmCBR3 after interference with BmSpr, the BH4 content did not change significantly. It is speculated that BmCBR3 is located upstream of BmSpr. These results provide a theoretical basis for in-depth exploration of the role of BmSDR in B. mori and also provide clues for the research of other animal-related diseases.

Carbonyl reduction of 4-oxonon-2-enal (4-ONE) by Sniffer from D. magna and D.pulex.

The α, ß-unsaturated aldehydes 4-oxonon-2-enal (4ONE) and 4-hydroxynon-2-enal (4HNE) are products of unsaturated fatty acids and ROS, and can be formed in lipid-rich tissues such as neurons. As strong electrophiles, both compounds react with DNA and proteins, and are capable of inactivating enzymes. However, both the human carbonyl reductase and the carbonyl reductase Drosophila melanogaster Sniffer are known to reduce 4ONE, a major lipid peroxidation product, to a less or non-toxic form. In this study, products formed during carbonyl reduction of 4ONE and 4HNE by recombinant Sniffer proteins from Daphnia magna and Daphnia pulex were investigated. A high-performance liquid chromatography analysis showed that Sniffer from D. magna converted 35.6% of 4ONE to 11.9% HNO and 23.7% 4HNE, while D. pulex converted 34.5% of this substrate to 14.8% HNO and 19.7% 4HNE. Thus, 4HNE is the main product formed from the sniffer-mediated reduction of 4ONE. The kinetic parameters obtained from the reduction of 4ONE were Km = 13.9 ± 2.1 µM, kcat = 1.53 s-1, kcat/km = 0.11 s-1 µM-1 for D. magna Sniffer and Km = 29.2 ± 4.3 µM, kcat = 0.64 s-1, kcat/km = 0.02 s-1 µM-1 for D. pulex Sniffer. These results demonstrate that Sniffer from D. magna and D. pulex are important enzymes involved in the carbonyl reductive biotransformation of 4ONE, a cytotoxic lipid peroxidation product. Noteworthy, the catalytic properties of both Daphnia Sniffer enzymes reflect previous findings with Sniffer from Drosophila melanogaster.

Influence of Clerodendrum volubile leaf extract on doxorubicin-induced toxicity and inhibition of carbonyl reductase mediated metabolism.

OBJECTIVES: Doxorubicin (DOX) is a commonly used chemotherapeutic drug. However, its non-target organ toxicities pose a serious problem. This study is to assess the protective role of Clerodendrum volubile leaf extract (CVE) against DOX-induced toxicities in rats. In addition, the inhibitory activities of three phytochemical compounds (Rutin, Gallic acid and Rosmarinic acid) from CVE against Carbonyl reductase 1 (CBR1) were examined. METHODS: Rats were randomly divided into 5 groups: (a) Control group rats were given 0.9% NaCl as vehicle, (b) DOX group: A single dose of DOX (25 mg/kg; i.p.) was administered and rats were sacrificed 4 days after DOX injection, while groups (c-e) CVE-treated DOX rat groups were given 125, 250 and 500 mg/kg body weight of extracts orally for 12 consecutive days; 8 days before, and 4 days after the DOX administration. Computational techniques were used to determine the inhibitory activities of the compounds against CBR1. RESULTS: DOX intoxication caused a significant increase (p<0.05) in serum marker enzymes: ALT, AST, ALP, LDH, CK activities. The levels of liver and heart tissues antioxidant parameters: GPx, SOD, CAT, and GSH were significantly (p<0.05) decreased in DOX-intoxicated rats with concomitant elevation of malondialdehyde levels. Pretreatment with CVE reversed the above trends. From the structural analysis, Rutin and RSA exhibited the highest binding free energies against CBR1, and also exhibited structural stability when bound with CBR1. CONCLUSIONS: Our study indicates the protective effect of CVE when used in combination with doxorubicin thus improving its chemotherapeutic application via inhibition of CBR-mediated metabolism.

Induction of carbonyl reductase 1 (CR1) gene expression in Daphnia magna by TNT, but not its key metabolites 2-ADNT and 4-ADNT.

2,4,6-trinitrotoluene (TNT) is a known source of reactive oxygen species (ROS), which cause oxidative stress in aquatic ecosystems. Carbonyl reductases (CRs) are one of several possible defense mechanisms induced against ROS products, especially those that result in the 'so-called' carbonyl stress. Daphnia magna, a freshwater organism living in stagnant freshwater bodies, expresses four copies of the CR gene (Dma_CR1, Dma_CR2, Dma_CR3 and Dma_CR4). In this study, induction of all four copies of Dma_CR by 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT), was investigated. Reverse transcription polymerase chain reaction (RT-PCR) analysis of treated daphnids revealed up-regulation of Dma_CR1 alone in response to TNT, but not 2-ADNT and 4-ADNT (which are key metabolites of TNT). This concentration- and time-dependent up-regulation in mRNA-expression was observed both in the presence and absence of light, in the same magnitude. Moreover, significant change in mRNA-expression could be observed 8 h after treatment with TNT. In the presence of TNT, the antioxidant N-acetylcysteine (NAc) could not reverse TNT-induced up-regulation of Dma_CR1 mRNA-expression. On the other hand, withdrawal of TNT from the culture medium caused a significant reduction in the TNT-induced mRNA-expression of Dma_CR1 within 24 h. These findings highlight the potential of Dma_CR1 as a biomarker for biomonitoring of TNT levels in freshwater bodies.

Chrysin induces autophagy-dependent ferroptosis to increase chemosensitivity to gemcitabine by targeting CBR1 in pancreatic cancer cells.

Recent studies have verified that inducing reactive oxygen species (ROS) is one of the gemcitabine anti-tumor mechanisms of action. Human carbonyl reductase 1 (CBR1) plays an important role in protecting cells against oxidative damage. However, it is unclear whether CBR1 is involved in pancreatic cancer (PC) progression and resistance to gemcitabine. Based on the GEPIA database, we analyzed tumor tissue samples from PC patients using immunohistochemistry (IHC) and revealed that CBR1 was highly expressed in PC tissues and that this was significantly correlated with the clinicopathological features of PC. Genetic inhibition of CBR1 suppressed PC cell proliferation by regulating ROS generation. Furthermore, gemcitabine upregulated CBR1 expression, which could limit the anti-tumor activity of gemcitabine, and attenuation of CBR1 enhanced gemcitabine sensitivity in vitro and in vivo. Additionally, we report that chrysin directly binds to CBR1, which inhibited its enzymatic activity both at the molecular and cellular levels. Inhibition of CBR1 by chrysin increased cellular ROS levels and led to ROS-dependent autophagy, which resulted in the degradation of ferritin heavy polypeptide 1 (FTH1) and an increase in the intracellular free iron level that participates in ferroptosis in PC cells. Finally, our results showed that chrysin enhanced PC sensitivity to gemcitabine by inducing ferroptotic death in vitro and in vivo. Collectively, these findings indicate that CBR1 is a potential therapeutic target for PC treatment. In addition, we elucidated a novel mechanism underlying the anti-tumor effects of chrysin.

Loxoprofen enhances intestinal barrier function via generation of its active metabolite by carbonyl reductase 1 in differentiated Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide as antipyretic analgesics and agents for rheumatoid arthritis and osteoarthritis, but known to cause damage to the gastrointestinal mucosae as their serious adverse effects. Few studies showed the impairment of intestinal epithelial barrier function (EBF) by high concentrations (0.5-1 mM) of NSAIDs, but the underlying mechanism is not fully understood. This study is aimed at clarifying effects at a low concentration (50 µM) of three NSAIDs, loxoprofen (Lox), ibuprofen and indomethacin, on intestinal EBF using human intestinal epithelial-like Caco-2 cells. Among those NSAIDs, Lox increased the transepithelial electric resistance (TER) value, decreased the paracellular Lucifer yellow CH (LYCH) permeability, and upregulated claudin (CLDN)-1, -3 and -5, indicating that low doses of Lox enhanced EBF through increasing expression of CLDNs. Lox is known to be metabolized to a pharmacologically active metabolite, (2S,1'R,2'S)-loxoprofen alcohol (Lox-RS), by carbonyl reductase 1 (CBR1), which is highly expressed in human intestine. CBR1 was expressed in the Caco-2 cells, and the pretreatment with a CBR1 inhibitor suppressed both the Lox-evoked CLDN upregulation and EBF enhancement. In addition, the treatment of the cells with Lox-RS resulted in higher TER value and lower LYCH permeability than those with Lox. Thus, Lox-RS synthesized by CBR1 may greatly contribute to the improving efficacy of Lox on the barrier function. Since EBF is decreased in inflammatory bowel disease, we finally examined the effect of Lox on EBF using the Caco-2/THP-1 co-culture system, which is used as an in vitro inflammatory bowel disease model. Lox significantly recovered EBF which was impaired by inflammatory cytokines secreted from THP-1 macrophages. These in vitro observations suggest that Lox enhances intestinal EBF, for which the metabolism of Lox to Lox-RS by CBR1 has an important role.

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase.

Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat /KM = 47.3 s-1 ·mM-1 ), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)-selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.

Maternal, umbilical arterial metabolic levels and placental Nrf2/CBR1 expression in pregnancies with and without 25-hydroxyvitamin D deficiency.

BACKGROUND: The aim of this case-control study was to document maternal, umbilical arterial metabolic levels and correlations in pregnancies with and without 25-hydroxyvitamin D [25(OH)D] deficiency, while, also investigating the expression of nuclear factor erythroid 2 related factor 2 (Nrf2) and carbonyl reductase 1 (CBR1) in the placenta. METHODS: One hundred participants, 50 deficient for 25(OH)D and 50 normal, were recruited from among hospitalized single-term pregnant women who had elected for cesarean section. Umbilical arterial and placental samples were collected during cesarean section. Metabolic levels were assessed for the 25(OH)D deficiency and control groups' maternal, umbilical arterial samples. Nrf2 and CBR1 expression levels were investigated in the placentas of 12 pregnant women with 25(OH)D deficiency and 12 controls. RESULTS: Compared with the control participants, the 25(OH)D deficient women had significantly higher triglyceride (TG) levels (3.80 ± 2.11 vs. 2.93 ± 1.16 mmol/L, 3.64 ± 1.84 vs. 2.81 ± 1.16 mmol/L, p < .01, .001); lower high density lipoprotein cholesterol (HDL-C) levels (1.54 ± 0.32 vs. 1.82 ± 0.63 mmol/L, 1.41 ± 0.72 vs. 2.44 ± 1.68 mmol/L, p < .001, .01) in both material blood and the umbilical artery. In addition, Nrf2 and CBR1 expression levels were lower in the maternal 25(OH)D deficient placenta. CONCLUSION: 25(OH)D deficient pregnant women have higher TG levels and lower HDL-C levels in both material blood and the umbilical artery. TG level is negatively correlated with 25(OH)D in both the maternal serum and infant umbilical artery. 25(OH)D deficiency also lowers placental expression of Nrf2 and CBR1.Supplemental data for this article is available online at here.

Carbonyl Reductase 1 Attenuates Ischemic Brain Injury by Reducing Oxidative Stress and Neuroinflammation.

Oxidative stress and neuroinflammatory response after the ischemic injury are important pathophysiologic mechanisms that cause brain tissue loss and neurological deficit. This study aims to observe the expression and role of carbonyl reductase 1 (CBR1), an NADPH-dependent oxidoreductase with specificity for carbonyl compounds such as 4-hydroxynonenal (4-HNE), in the brain after ischemic injury and to investigate the influence of CBR1 on ischemia-induced neuroinflammation. CBR1 expresses in the neurons, astrocyte, and microglia in the normal brain. The expression of CBR1 decreased in the ischemic regions following cerebral ischemia, and also reduced in primary neurons after OGD (oxygen-glucose deprivation); however, the expression of CBR1 significantly increased in microglia in the ischemic penumbra. Furthermore, TAT-CBR1 fusion protein played neuroprotective effects in reducing the infarct volume and improving neurological outcomes after ischemic injury. Mechanistically, CBR1 decreased the levels of 4-HNE in the brain after stroke; it also modulated microglial polarization toward the M2 phenotype, which was well-known to confer neuroprotection after ischemic injury. Our results demonstrate that CBR1 provides neuroprotection against ischemic injury by reducing oxidative stress and neuroinflammation, making a promising agent for cerebral ischemia treatment.