Carbonyl Reductase Research Articles

Metabolic Dysfunction-Associated Steatotic Liver Disease Is Accompanied by Increased Activities of Superoxide Dismutase, Catalase, and Carbonyl Reductase 1 and Levels of miR-200b-3p in Mouse Models

Metabolic dysfunction-associated steatotic liver disease (MASLD), one of the leading causes of chronic liver disorders, is characterized by hepatic lipid accumulation. MASLD causes alterations in the antioxidant defense system, lipid, and drug metabolism, resulting in impaired antioxidant status, hepatic metabolic processes, and clearance of therapeutic drugs, respectively. In the MASLD pathogenesis, dysregulated epigenetic mechanisms (e.g., histone modifications, DNA methylation, microRNAs) play a substantial role. In this study, the development of MASLD was investigated in mice fed a high-fat, high-fructose, and high-cholesterol (FFC) diet from 2 months of age, mice treated neonatally with monosodium glutamate (MSG) on a standard diet (STD), and mice treated with MSG on an FFC diet at 7 months of age and compared to control mice (C) on STD. Changes in liver histology, detoxification enzymes, epigenetic regulation, and genes involved in lipid metabolism were characterized and compared. The strong liver steatosis was observed in MSG STD, C FFC, and MSG FFC, with significant fibrosis in the latter one. Moreover, substantial alterations in hepatic lipid metabolism, epigenetic regulatory factors, and expressions and activities of various detoxification enzymes (namely superoxide dismutase, catalase, and carbonyl reductase 1) were observed in MASLD mice compared to control mice. miR-200b-3p, highly significantly upregulated in both FFC groups, could be considered as a potential diagnostic marker of MASLD. The MSG mice fed FFC seem to be a suitable model of MASLD characterized by both liver steatosis and fibrosis and substantial metabolic dysregulation.

Improving the catalytic performance of carbonyl reductase based on the functional loops engineering.

Vibegron functions as a potent and selective β3-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (kcat/Km = 260.3 s-1 mM-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.

Highly efficient synthesis of the chiral ACE inhibitor intermediate (R)-2-hydroxy-4-phenylbutyrate ethyl ester via engineered bi-enzyme coupled systems

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester ((R)-HPBE) is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), catalyzed by carbonyl reductase (CpCR), with efficient cofactor regeneration playing a crucial role. In this study, an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase (CpCR) with glucose dehydrogenase (GDH), resulting in the construction of five recombinant strains capable of NADPH regeneration. Among these, the recombinant strain E. coli BL21-pETDuet-1-GDH-L-CpCR, where CpCR is fused to the C-terminus of GDH, demonstrated the highest catalytic activity. This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%, with an enantiomeric excess (ee) of 99.9% during the conversion of 30 mM OPBE to (R)-HPBE. High-density fermentation further enhanced enzyme yield, achieving an enzyme activity of 1960 U/mL in the fermentation broth, which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation. Additionally, the implementation of a substrate feeding strategy enabled continuous processing, allowing the strain to efficiently convert a final OPBE concentration of 920 mM, producing 912 mM of (R)-HPBE. These findings highlight the system’s improved catalytic efficiency, stability, and scalability, making it highly suitable for industrial-scale biocatalytic production.

A quantitative proteomic approach to evaluate the efficacy of carnosine in a murine model of chronic obstructive pulmonary disease (COPD)

The aim of the work was to study a dose-dependent effect of inhaled carnosine (10, 50 or 100 mg/kg/day) in mice exposed to cigarette smoke as a model of chronic obstructive pulmonary disease (COPD). A dose-dependent loading of the dipeptide in lung tissue and bronchoalveolar lavage (BAL) was firstly demonstrated by LC-ESI-MS analysis. Cigarette smoke exposure induced a significant lung inflammation and oxidative stress in mice which was dose-dependently reduced by carnosine. Inflammation was firstly evaluated by measuring the cytokines content in the BAL. All the measured cytokines were found significantly higher in the smoke group in respect to control, although the data are affected by a significant variability. Carnosine was found effective only at the highest dose tested and significantly only for keratinocyte-derived cytokine (KC). Due to the high variability of cytokines, a quantitative proteomic approach to better understand the functional effect of carnosine and its molecular mechanisms was used. Proteomic data clearly indicate that smoke exposure had a great impact on lung tissue with 692 proteins differentially expressed above a threshold of 1.5-fold. Protein network analysis identified the activation of some pathways characteristic of COPD, including inflammatory response, fibrosis, induction of immune system by infiltration and migration of leukocyte pathways, altered pathway of calcium metabolism and oxidative stress. Carnosine at the tested dose of 100 mg/kg was found effective in reverting all the pathways evoked by smoke. Only a partial reverse of the dysregulated proteins was evident at low- and mid-tested doses, although, for some specific proteins, indicating an overall dose-dependent effect. Regarding the molecular mechanisms involved, we found that carnosine upregulated some key enzymes related to Nrf2 activation and in particular glutathione peroxidase, reductase, transferase, SOD, thioredoxins, and carbonyl reductase. Such mechanism would explain the antioxidant and anti-inflammatory effects of the dipeptide.

Gene hunting and semi-rational design of carbonyl reductase from Kosakonia radicincitans for highly efficient synthesis of the key chiral intermediate of Telotristat ethyl

Carbonyl reductase exhibits significant potential in the asymmetric production of chiral alcohols. (αR)-4-Chloro-2-(3-methyl-1H-pyrazol-1-yl)-α-(trifluoromethyl)benzenemethanol ((R)-CMPPFO) is a critical precursor for the synthesis of Telotristat ethyl, an oral drug for the treatment of diarrhea in carcinoid syndrome. Herein, a novel carbonyl reductase KrSDR5 from Kosakonia radicincitans was obtained using gene hunting strategy, capable of asymmetrically reducing the precursor ketone 1-[4‑chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanone (CMPPFA) to (R)-CMPPFO with strict R-stereoselectivity (>99.9 % ee). Further, semi-rational design was adopted to acquire a positive mutant KrSDR5T91V/V141M/I159V, with assistance from a comparative analysis of enzyme-substrate binding mode in molecular dynamics (MD) simulations. This variant displayed a 12.4-fold increase in kcat/Km towards CMPPFA compared to the wild-type (WT) KrSDR5. Insights were gained on the high enantioselectivity and the enhancement of enzyme catalytic activity of the mutant through MD simulations. Using the whole-cells of KrSDR5T91V/V141M/I159V as biocatalyst, the asymmetric synthesis of (R)-CMPPFO was achieved within 20 h at 500 mM CMPPFA concentration, resulting in a 95.0 % yield with >99.9 % ee, and a highest space-time yield (STY) of 165.7 g·L-1·d-1 compared with previous reports. This study provides a robust biocatalyst for highly efficient production of the key precursor (R)-CMPPFO for Telotristat ethyl, highlighting its potential in the biosynthesis of pharmaceutical intermediates.

Adipocyte maturation impacts daunorubicin disposition and metabolism.

Acute lymphoblastic leukaemia (ALL) is the most common type of childhood leukaemia with effective chemotherapeutic treatment. However, obesity has been associated with higher ALL chemoresistance rates and lower event-free survival rates. The molecular mechanism of how obesity promotes chemotherapy resistance is not well delineated. This study evaluated the effect of adipocyte maturation on sequestration and metabolism of chemotherapeutic drug daunorubicin (DNR). Using targeted LC-MS/MS multi-analyte assay, DNR sequestration and metabolism were studied in human preadipocyte and adipocyte cell lines, where expressions of DNR-metabolizing enzymes aldo-keto reductases (AKR) and carbonyl reductases (CBR) were also evaluated. In addition, to identify the most DNR-metabolizing AKR/CBR isoforms, recombinant human AKR and CBR enzymes were subject to DNR metabolism. The results were further validated by AKR-, CBR-specific inhibitors. This report shows that adipocyte maturation upregulates expressions of AKR and CBR enzymes (by 4- to 60- folds, p < .05), which is positively associated with enhanced sequestration and metabolism of DNR in adipocytes compared to preadipocytes (by ~30%, p < .05). In particular, adipocyte maturation upregulates AKR1C3 and CBR1, which are the predominate metabolic enzyme isoforms responsible for DNR biotransformation to its metabolites. Fat is an expandable tissue that can sequester and detoxify DNR when stimulated by obesity, likely through the upregulation of DNR-metabolizing enzymes AKR1C3 and CBR1. Our data partially explains why obese ALL patients may be more likely to become chemoresistant towards DNR, and provides evidence for potential clinical investigation targeting obesity to reduce DNR chemoresistance.

Lipid Hydrolysis, Oxidation, and Fatty Acid Formation Pathway Mapping of Synergistically Fermented Sausage and Characterization of Lipid Mediating Genes.

Starter cultures play a significant role in lipid hydrolysis, prevention of lipid oxidation, and synthesis of fatty acid in fermented sausage, enhancing product quality. In this study, five synergistic bacterial strains were used, including Pediococcus pentosaceus (B-3), Latilactobacillus sakei DLS-24 (D-24), Latilactobacillus acidophilus DLS-29 (D-29), Lactiplantibacillus pentosus (B-1), and Lactiplantibacillus plantarum (B-2). Sausage B1B3D24 gave the highest free fatty acid with 39.45 g/100 g at 45-Day. Based on 2-thiobarbituric acid reactive substance, B2B3 contains 112.68 MDA/kg. Lipoxygenase activity displays the lowest in B1B3D24 with 0.095 μmol/min·mg followed by B2B3 with 0.145 μmol/min·mg. B1B3D24 contains 11.35 g/kg of monounsaturated fatty acid with the highest content in eicosenoic acid (C20:1) and palmitoleic acid (C16:1). The fatty acid synthesis pathway in B1B3D24 contains an active positive interaction with PUFA to increase the isotopomers of ω-3 and ω-6 fatty acids. In addition, lipid mediating genes in B1B3D24 show the highest counts in fatty-acid synthase, carbonyl reductase 4, 3-oxoacyl-[acyl-carrier-protein] synthase III, hydroxysteroid 17-beta dehydrogenase 8, and acetyl-CoA carboxylase.

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 11β-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/β-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3β-HSD and 3α-HSD, respectively). Additionally, 17β-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/β-dihydro-C21-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5β-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/β-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/β-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/β,17β-HSDs.

Rational redesign of the loop dynamics of carbonyl reductase LfSDR1 to improve the stereoselectivity for asymmetric synthesis of bulky chiral alcohols

Engineering biocatalysts with enhanced stereoselectivity is highly desirable, and active-site loop dynamics play an important role in its regulation. However, knowledge of their precise roles in catalysis and evolution is limited. Here, we used the strategy of Rosetta enzyme design combined molecular dynamic simulations (MDs) to reprogram the landscapes of the key active-site loop dynamics of the carbonyl reductase LfSDR1 to improve stereoselectivity. The key flexible loop in the active site showed the potential to regulate the catalytic properties. A library of virtual variants was produced using the Rosetta design and assessed dynamic effect of the loop with the aid of MDs. A potential candidate was obtained with significant stereoselectivity (ee > 99 %) compared to the wild-type (ee = 42 %) without loss of catalytic activity or thermostability. The molecular basis of the catalytic property enhancement was flanked by MDs, which revealed the role of the G92L mutation in regulating loop dynamics to stabilize the environment of the active site. Finally, a series of the challenge bulky substrate derivatives were assessed using the G92L variant, and all showed improved stereoselectivity ee > 99 %. This study provides novel insights for improving stereoselectivity through rational engineering of the loop dynamics of biocatalysts.

Overexpression of carbonyl reductase 1 in ovarian cancer cells suppresses proliferation and activates the eIF2 signaling pathway.

High expression of carbonyl reductase 1 (CBR1) protein in ovarian cancer cells inhibits tumor growth and metastasis. However, the underlying mechanism is unknown. To investigate the mechanism by which CBR1 suppresses tumor growth, the present study generated ovarian cancer cells that constitutively overexpress human CBR1 (hCBR1) protein. Ovarian cancer cell lines (OVCAR-3 and SK-OV-3) were transfected with a plasmid encoding hCBR1, followed by selection with G418 to isolate hCBR1-overexpressing lines. The proliferation rates of hCBR1-overexpressing cells were then compared with those of negative control and wild-type cells. Overexpression of hCBR1 led to significant inhibition of proliferation (P<0.05). Subsequently, to investigate changes in intracellular signaling pathways, cellular proteins were extracted and subjected to proteome analysis using liquid chromatography followed by mass spectrometry. There was an inverse correlation between CBR1 protein expression and cell proliferation. In addition, Ingenuity Pathway Analysis of hCBR1-overexpressing cell lines was performed, which revealed changes in the expression of proteins involved in signaling pathways related to growth regulation. Of these, the eukaryotic translation initiation factor 2 (eIF2) signaling pathway was upregulated most prominently. Thus, alterations in multiple tumor-related signaling pathways, including eIF2 signaling, may lead to growth suppression. Taken together, the present data may lead to the development of new drugs that target CBR1 and related signaling pathways, thereby improving outcomes for patients with ovarian cancer.

Catalytic Synthesis of (S)-CHBE by Directional Coupling and Immobilization of Carbonyl Reductase and Glucose Dehydrogenase.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.

Dual enzyme self-assembly cluster for high efficient asymmetric reduction of ethyl 6-oxo-8-chlorooctanoate

(R)-ECHO is synthesized through the asymmetric reduction of ethyl 6-oxo-8-chlorooctanoate (ECOO) using carbonyl reductase(CR2) as a catalyst and co-enzyme NADPH as a hydrogen donor with addition of glucose dehydrogenase (GDH) for NADPH regeneration, thereby enhancing biotransformation efficiency. To improve the reaction efficiency further, a dual enzyme self-assembly cluster EutM-SC-ST-CR2(GDH) was developed specifically for this purpose. This co-immobilised system demonstrated a 50% increase in the conversion of ECOO compared to when SpyTag-CR2(ST-CR2) and SpyTag-GDH(ST-GDH) were co-catalysts. The optimal conditions for this increase were observed to be at a pH of 8.0, 30°C, when concentrations of 250 mM ECOO, 270 mM glucose and 0.1 mM NADPH, and a molar ratio of ST-CR2 to ST-GDH to EutM-SpyCatcher(EutM-SC) of 1:1:2 (molecular concentrations of ST-CR2, ST-GDH and EutM-SC were 3.5 mM, 3.5 mM and 7 mM). Under these conditions, the conversion of ECOO reached up to 95% and the enantiomeric excess (e.e.) of (R)-ECHO achieved 99.9%. Moreover, the EutM-SC-ST-CR2(GDH) construct displayed enhanced enzyme activity, stability, and kinetics, as well as improvements in electrostatic and ionic strengths. In conclusion, this study presents the development of a novel self-assembled protein scaffold, EutM-SC, for the co-immobilisation of ST-CR2 and ST-GDH, thus optimizing the biological reaction conditions and offering potential for the industrial production of (R)-α-lipoic acid.

Integrated engineering at distal site and active center regulates stereoselectivity and activity of carbonyl reductase towards N-Boc-pyrrolidone

Green and efficient biosynthesis of two enantiomeric products of N-Boc-pyrrolidinol (NPBL) as key pharmaceutical blocks for treatment of cancer and HIV has received much attention. The precursor, N-Boc-pyrrolidone (NPBO), is considered as a difficult-to-reduce ketone by carbonyl reductases, because it has a bulky Boc group and sterically similar substitutions on either side of the carbonyl group resulting in low stereoselectivity and conversion. Moreover, activity enhancement concomitant with reversal of stereoselectivity is challenging. Carbonyl reductase (CpCR) from Candida parapsilosis shows 90 %ee for (S)-NBPL, but low catalytic efficiency. In this study, we considered both activity and selectivity, and proposed an integrated engineering strategy. The distal site was introduced from an activity perspective and the active pocket sites were introduced by virtual saturation mutagenesis from a stereoselectivity perspective. Mutants L34A/W116A (95 %ee (S)) and L34A/W116T/F285C/W286S (94 %ee (R)) were obtained with 26.8-fold and 2.9-fold higher catalytic efficiencies, respectively. Molecular dynamics simulations revealed the mechanism of stereoselective flip-flop and activity enhancement; mutation altered the substrate-binding mode and changed the shape and size of the cavities, thus contributing to the change in the ratio of active conformations in the pre-reactive state. This study, which differs from the traditional method of exchanging large and small pockets, provides a viable approach for the rational design of carbonyl reductases with high stereoselectivity for target substrates.

Using isopropanol as the hydrogen donor to achieve by-product-free biological hydrogenation in water with carbonyl reductase

In this study, the reaction equilibrium was broken using isopropanol as a co-substrate, which was different from previous glucose as a co-substrate for furfural reduction, solving the problems of many by-products, low selectivity, and the addition of sodium phosphate buffers. The volatility of isopropanol and acetone prevented the production of by-products to the reaction system, improving the conversion and selectivity. 10 g/L furfural could be converted into furfuryl alcohol in pure water, and the furfural conversion was 90%, furfuryl alcohol selectivity was 100%, and the concentration of furfuryl alcohol could reach more than 20 g/L through accumulation reaction. No by-products were produced in this reaction system, and a single furfuryl alcohol product was obtained in the water catalytic system, which was convenient for downstream separation and purification. At the same time, based on the theory of the NADH coenzyme cycle system, this catalytic method could complete the conversion of various aldehydes into the corresponding alcohols, and it could be expanded to various biological hydrogenation projects, which has important industrial development and production prospects.

Identification and two-step tunnel engineering of a carbonyl reductase for biosynthesis of an (R)-α-lipoic acid intermediate

As a “universal antioxidant”, α-lipoic acid (LA) is beneficial for health and therapy, combatting Alzheimer's disease, diabetes. The main active form of LA has the R-configuration. Biocatalytic synthesis of (R)-ethyl 8‑chloro-6-hydroxycaprylate ((R)-ECHO) from ethyl 8‑chloro-6-oxocrylate (ECOO) coupled with subsequent chemical synthesis is a competitive route to (R)-LA. However, very few enzymes have been confirmed to prepare the key intermediate (R)-ECHO with high efficiency and absolute stereoselectivity. In this work, carbonyl reductase CoCR13 from Candida orthopsilosis Co 90–125 was mined, and then engineered by a stepwise strategy with the aim of expanding the substrate entry tunnel. This led to the optimal variant M3 (R129L/D210T/S126A), which exhibited a 93.42-fold higher catalytic efficiency (kcat/Km = 85.95 mm−1·s−1) towards ECOO than the wild type, an ee value greater than 99.5 % (R) from initial 86 % (R), and a space time yield of 99.9 g·L−1·h−1. This study expands the scarce enzyme inventory for the biosynthesis of (R)-ECHO and offers a new choice for the industrial preparation of (R)-LA through a synergistic enzymatic-chemical approach.

The cardioprotective properties of Persicaria maculosa and Citrus sinensis extracts against doxorubicin-induced cardiotoxicity in mice.

This study assessed the cardioprotective properties of Persicaria maculosa (PME) and Citrus sinensis (CME) hydro-methanolic extracts, besides Citrus sinensis aqueous extract (CWE) against doxorubicin (DOX)-induced cardiotoxicity. The extracts were characterized. Mice were divided into eight groups: control (saline), DOX, protected (injected with 200 mg/kg of PME, CWE or CME for 21 days, orally, and DOX), and extracts (PME, CWE or CME administration, orally, for 21 days). DOX was injected (5 mg/kg, ip) on days 8, 13 and 18 of the experiment. Cardiac tumor necrosis factor-alpha (TNF-α), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and carbonyl reductase 1 (CBR1) expression levels, besides superoxide dismutase, catalase, malondialdehyde, nitric oxide and total protein levels were evaluated. Serum lactate dehydrogenase, creatine phosphokinase cardiac isoenzyme, aspartate transaminase, cholesterol, triglycerides and creatinine levels, as well as the cardiac tissues were examined. Comparing with the control, DOX considerably (p<0.01) up-regulated TNF-α expression, malondialdehyde, nitric oxide, cardiac enzymes, lipids and creatinine levels, while it significantly (p<0.01) down-regulated Nrf2 and CBR1. Additionally, DOX interfered with antioxidant enzymes' activities (p<0.01). Conversely, protected groups showed a significant (p<0.01) amelioration of DOX-induced cardiotoxic effects. The current study provides a new understanding of P. maculosa and C. sinensis cardioprotective mechanisms. The extracts' cardioprotective effects may be due to their antioxidant activities, ability to maintain the redox homeostasis through regulation of important antioxidant genes and primary antioxidant enzymes, and capability to recover inflammatory cytokines and lipids levels. Noteworthy, the tested extracts showed no toxic changes on the normal mice.

Based on mRNA Sequencing Techniques to Explore the Molecular Mechanism of Buzhong Yiqi Decoction for Autoimmune Thyroiditis.

Autoimmune diseases (AD) account for a high percentage of the population. One of the most prevalent is autoimmune thyroiditis (AIT). However, the therapeutic effects of Buzhong Yiqi (BZYQ) decoction on AIT have not been studied yet. The majority of the present study was conducted on NOD.H-2h4 mice in an attempt to ascertain the therapeutic effects of BZYQ decoction on AIT. The 0.05% sodium iodide water (NaI)-induced AIT mice model was established. A total of nine NOD.H-2h4 mice were randomly divided into three groups: the normal group provided with regular water, the model group drinking freely 0.05% NaI, and the treatment group treated with BZYQ decoction (9.56 g/kg) after NaI supplementation (NaI + BZYQ). BZYQ decoction was administered orally once daily for eight weeks. The thyroid histopathology test was used to measure the severity of lymphocytic infiltration. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of anti-thyroglobulin antibody (TgAb), interleukin (IL)-1β, IL-6, and IL-17. The Illumina HiSeq X sequencing platform was utilized to analyze the thyroid tissue by mRNA expression profiles. Bioinformatics analysis was used to investigate the biological function of the differentially expressed mRNAs. In addition, the expression of Carbonyl Reductase 1 (CBR1), 6-Pyruvoyltetrahydropterin Synthase (PTS), Major Histocompatibility Complex, Class II (H2-EB1), Interleukin 23 Subunit Alpha (IL-23A), Interleukin 6 Receptor (IL-6RA), and Janus Kinase 1 (JAK1) was measured by quantitative real-time PCR (qRT-PCR). The treatment group exhibited significantly lower rates of thyroiditis and lymphocyte infiltration compared to the model group. Serum levels of TgAb, IL-1β, IL-6, and IL-17 were significantly higher in the model group, but they fell dramatically after BZYQ decoction administration. According to our results, 495 genes showed differential expression in the model group compared to the control group. Six hundred twenty-five genes were significantly deregulated in the treatment group compared to the model group. Bioinformatic analysis showed that most mRNAs were associated with immune-inflammatory responses and were involved in multiple signaling pathways, including folate biosynthesis and the Th17 cell differentiation pathway. CBR1, PTS, H2-EB1, IL- 23A, IL-6RA and JAK1 mRNA participated in folate biosynthesis and the Th17 cell differentiation pathway. The qRT-PCR analysis confirmed that the above mRNAs were regulated in the model group compared to the treatment group Conclusion: The results of this investigation have revealed novel insights into the molecular mechanism of action of BZYQ decoction against AIT. The mechanism may be partially attributed to the regulation of mRNA expression and pathways.

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.

In vitro evaluation of the reductive carbonyl idarubicin metabolism to evaluate inhibitors of the formation of cardiotoxic idarubicinol via carbonyl and aldo–keto reductases

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo–keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.