Chemoenzymatic total synthesis of rotigotine via IRED-catalyzed reductive amination.

A short and chemoenzymatic synthesis of rotigotine using an IR-36-M5 mutant is reported. Focusing on the residues that directly contact the 2-tetralone moiety, we applied structure-guided semi-rational design to obtain a double-mutant F260W/M147Y, which showed a good isolated yield and S-stereoselectivity >99% toward 2-aminotetralin synthesis. Furthermore, the utility of this biocatalytic protocol was successfully demonstrated in the enantioselective synthesis of rotigotine via enzymatic reductive amination as the key step.

Semi-rational design of an imine reductase for asymmetric synthesis of alkylated S-4-azepanamines.

Although imine reductase (IRED)-catalyzed reductive amination is promising for the synthesis of alkylated chiral amines, precisely regulating the stereoselectivity of IRED remains a great challenge. Herein, focusing on the residues directly in contact with the ketone moiety, we applied structure-guided semi-rational design to obtain the triple-mutant I149Y/L200H/W234K. This mutant showed high stereoselectivity, of up to >99% (S), toward reductive amination of N-Boc-4-oxo-azepane and different amines, and to the best of our knowledge is the first biocatalyst developed for asymmetric synthesis of chiral azepane-4-amines.

Structure-guided semi-rational design of an imine reductase for enantio-complementary synthesis of pyrrolidinamine.

In this study, engineered imine reductases (IREDs) of IRED M5, originally from Actinoalloteichus hymeniacidonis, were obtained through structure-guided semi-rational design. By focusing on mutagenesis of the residues that directly interact with the ketone donor moiety, we identified two residues W234 and F260, playing essential roles in enhancing and reversing the stereoselectivity, respectively. Moreover, two completely enantio-complementary variants S241L/F260N (R-selectivity up to 99%) and I149D/W234I (S-selectivity up to 99%) were achieved. Both variants showed excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing pyrrolidinamines. Its application was demonstrated in a short synthesis of the key intermediates of potential drug molecules leniolisib and JAK1 inhibitor 4, from cheap and commercially available pro-chiral N-Boc-piperidone 1 (2 and 3 steps, respectively).

Plasma Angiopoietin-2 as a Promising Biomarker for the Prognosis of Acute Pulmonary Embolism.

BACKGROUND: Endothelial damage is one of the pathogenic conditions of acute pulmonary embolism (APE). The essential role of angiogenin, ribonuclease A family, member 2 (Ang-2) in APE remains unclear. This study aimed to investigate the predictive value of Ang-2 in the clinical outcomes of patients with APE. METHODS: Plasma Ang-2 levels were measured by an enzyme-linked immunosorbent assay kit using a DuoSet methodology in 118 APE patients and 53 healthy controls. Baseline data relevant to mortality over time were obtained from hospital databases or by patient's follow-up (median follow-up time: 25.0 ± 13.2 months). The main outcome was all-cause mortality. RESULTS: Plasma Ang-2 level was significantly higher in APE patients than in healthy controls (p < 0.001). Patients dying during the first 30 days presented higher baseline levels of Ang-2 than the survivors (p < 0.001). Patients dying during the follow-up also showed higher baseline levels of Ang-2 than the survivors (p < 0.001). The multi-variable logistic regression analysis showed that the N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) [odds ratio (OR): 19.8; 95% CI: 1.5 - 255.8; p = 0.022] and Ang-2 (OR: 9.9; 95% CI: 1.4 - 70.5; p = 0.022) emerged as independent predictors of the 30-day mortality. Furthermore, the multivariable Cox's regression identified plasma Ang-2 [hazards ratio (HR): 1.35; 95% CI: 1.10 - 1.66; p = 0.004] as an independent predictor of long-term mortality in patients with APE. CONCLUSIONS: A high circulating level of Ang-2 can be considered as an independent predictor for the poor outcome of APE and may serve as a biomarker for the risk stratification in patients with APE.

Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering.

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm , and a significant increase in the e.e. value from 78 % (R) to >99 % (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.

Effects of 27 CYP3A4 protein variants on saxagliptin metabolism in vitro.

Saxagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor widely used in patients with type 2 diabetes. It can increase the amount of insulin after meals and lower blood sugar. CYP450 3A4 (CYP3A4) can metabolize about 30%-40% of therapeutic drugs. Individual differences caused by CYP3A4 genetic polymorphisms can lead to treatment failure, unpredictable side effects, or severe drug toxicity. The aim of this study was to evaluate the catalytic activities of 27 CYP3A4 variants on saxagliptin metabolism in vitro, which were identified in human CYP alleles. We successfully constructed 27 kinds of wild-type and variant vectors of pFast-dual-OR-3A4 by overlap extension PCR and prepared 27 kinds of CYP3A4 highly expressed cell microsomes by baculovirus insect cell expression system. The ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to detect the concentrations of the metabolite of saxagliptin (5-hydroxysaxagliptin) and the internal standard. Compared with the wild-type CYP3A4.1, the intrinsic clearance values of most varieties decreased to 1.91%-77.08%. Most of these varieties showed a decrease in Vmax and an increase in Km values compared with wild type. We are the first to report the vitro metabolic data of 27 CYP3A4 variants of the metabolism of saxagliptin which can deepen our understanding of individualized drug use by combining previous studies about the effects of CYP3A4 variants of drug metabolism. With further in vivo studies, we hope it can guide individualized drug use in the clinic when the variants with low metabolic activity to saxagliptin were sequenced in the human body.

Actinomycetes-derived imine reductases with a preference towards bulky amine substrates.

Since imine reductases (IREDs) were reported to catalyze the reductive amination reactions, they became particularly attractive for producing chiral amines. Though diverse ketones and aldehydes have been proved to be excellent substrates of IREDs, bulky amines have been rarely transformed. Here we report the usage of an Increasing-Molecule-Volume-Screening to identify a group of IREDs (IR-G02, 21, and 35) competent for accepting bulky amine substrates. IR-G02 shows an excellent substrate scope, which is applied to synthesize over 135 amine molecules as well as a range of APIs' substructures. The crystal structure of IR-G02 reveals the determinants for altering the substrate preference. Finally, we demonstrate a gram-scale synthesis of an analogue of the API sensipar via a kinetic resolution approach, which displays ee >99%, total turnover numbers of up to 2087, and space time yield up to 18.10 g L-1 d-1.

A hybrid system for the overproduction of complex ergot alkaloid chanoclavine.

Synthetic biology-based methods (Sbio) and chemical synthesis (Csyn) are two independent approaches that are both widely used for synthesizing biomolecules. In the current study, two systems were combined for the overproduction of chanoclavine (CC), a structurally complex ergot alkaloid. The whole synthetic pathway for CC was split into three sections: enzymatic synthesis of 4-Br-Trp (4-Bromo-trptophan) using cell-lysate catalysis (CLC), chemical synthesis of prechanoclavine (PCC) from 4-Br-Trp, and overproduction CC from PCC using a whole-cell catalysis (WCC) platform. The final titer of the CC is over 3 g/L in this Sbio-Csyn hybrid system, the highest yield reported so far, to the best of our knowledge. The development of such a combined route could potentially avoid the limitations of both Sbio and Csyn systems and boost the overproduction of complex natural products.

Urinary 8-oxoGuo as a potential novel evaluation index for patients with nephrotic syndrome.

Urinary 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'- deoxyguanosine (8-oxodGuo) are considered biomarkers of oxidative stress, and patients with nephrotic syndrome have been reported to have increased oxidative stress levels. In this study, we aimed to assess the value of 8-oxoGuo and 8-oxodGuo as novel biomarkers to evaluate the severity of nephrotic syndrome. In total, 107 patients with nephrotic syndrome and 116 healthy controls were recruited for this study. The concentrations of urinary 8-oxoGuo and 8-oxodGuo were measured using isotope-labeled liquid chromatography with tandem mass spectrometry. Urinary creatinine was used to regulate 8-oxoGuo and 8-oxodGuo concentrations. Urinary 8-oxoGuo and 8-oxoGuo/Cr levels in patients with nephrotic syndrome were significantly higher than those in healthy control participants. 8-oxoGuo/Cr showed a positive correlation with the 24 h urinary total protein (UTP) and UTP levels and negative correlations with serum total protein and albumin levels. After treatment, urinary 8-oxoGuo and 8-oxoGuo/Cr levels were significantly lower in the group with a low 24 h-UTP value (<3.5 g/d) than in the high value group. 8-oxoGuo can be used as a feasible and reliable biomarker for the assessment of nephrotic syndrome.HighlightsUrinary 8-oxoGuo level was significantly increased in patients with nephrotic syndrome.Urinary 8-oxoGuo level increased with an increase in plasma protein and a decrease in urine protein.Urinary 8-oxoGuo level decreased with nephrotic syndrome remission when urinary microalbumin showed no significant change.Urinary 8-oxoGuo level can be used as novel biomarkers of nephrotic syndrome.

Effects of 31 recombinant CYP2C19 variants on clomipramine metabolism in vitro.

BACKGROUND: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31CYP2C19 alleles in the Han Chinese population; studying the effects of CYP2C19 on drug metabolism can help reduce adverse drug reactions and therapeutic failure. AIM: The aim of this study was to assess the catalytic activities of 31 allelic isoforms and their effects on the metabolism of clomipramine in vitro. METHODS: The wild-type and 30 CYP2C19 variants were expressed in insect cells, and each variant was characterized using clomipramine as the substrate. Reactions were performed at 37°C with 5-150 µmol/L substrate for 30 min. By using ultra-high-performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl clomipramine were determined. RESULTS: Among the CYP2C19 variants tested, CYP2C19*29, L16F, and T130M showed extremely increased intrinsic clearance of clomipramine, CYP2C19*3C, and N277K showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 0.65% to 63.28%). In addition, CYP2C19*3 and 35FS could not be detected because they have no detectable enzyme activity. CONCLUSIONS: As the first report of 31 CYP2C19 alleles for clomipramine metabolism, our study could provide corresponding reference for clomipramine for further studies in vivo and offer valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus-insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

Systemic RNA oxidation can be used as a biomarker of infection in challenged with Vibrio parahaemolyticus.

More and more evidence support the concept that RNA oxidation plays a substantial role in the progress of multiple diseases; however, only a few studies have reported RNA oxidation caused by microbial pathogens. Urinary 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn), which are broadly used as indicators of oxidative damage of RNA and DNA, were analyzed in this study to determine which can be used as a biomarker of infection in challenged with Vibrio parahaemolyticus (V. parahaemolyticus). In this work, 24 specific-pathogen-free (SPF) male SD rats were randomly divided into two groups: an infection group and a phosphate-buffered saline (PBS) control group. Our results proved that 8-oxo-Gsn rather than 8-oxo-dGsn was significantly increased after challenged with V. parahaemolyticus in urine and tissue samples of SD rats compared with the PBS control group. Simultaneously, white blood cells (WBCs) counts, intestinal inflammation and inflammatory factors (including CRP, IL-6, IL-1ß, TNF-α, IL-10, and IL-17A) were also increased sharply. Which has more clinical value is that the trend of urinary 8-oxo-Gsn was consistent with WBCs, intestinal inflammation and all kinds of inflammatory factors. More importantly is that urinary 8-oxo-Gsn of infection group was positively correlated with WBCs and various inflammatory cytokines. In a word, our results demonstrated that as a systemic RNA oxidation biomarker, we hope 8-oxo-Gsn can be used as a biomarker of the severity of microbial pathogens infection, rather than a specific biomarker of microbial pathogens infection.

Targeted elimination of intracellular reactive oxygen species using nanoparticle-like chitosan- superoxide dismutase conjugate for treatment of monoiodoacetate-induced osteoarthritis.

In our previous work, cationic functionalized chitosan was chemically conjugated with superoxide dismutase (SOD) to yield a unique nanoparticle-like conjugate O-HTCC-SOD that has demonstrated superior potential in treating reactive oxygen species (ROS)-related disorders to SOD. Considering contribution of ROS to pathogenesis of osteoarthritis, O-HTCC-SOD was firstly measured for effect on rat chondrocytes exposure to monoiodoacetate (MIA). O-HTCC-SOD was nontoxic to chondrocytes and had more long-acting and intracellular protection effects on chondrocytes against MIA-induced oxidative damage due to its superior elimination of intracellular ROS to SOD. Pharmacokinetic analysis demonstrated that O-HTCC-conjugated SOD significantly prolonged half-life and residence in rat joint cavity, and improved bioavailability compared with unmodified SOD. Intra-articular injection of O-HTCC-SOD significantly attenuated mechanical allodynia in MIA-induced osteoarthritis rats, dramatically suppressed gross morphological and histological lesions of articular cartilage, and greatly enhanced in vivo antioxidant capacity and anti-inflammatory effect. But native SOD had no obvious therapeutic effects. Consequently, the nanoparticle-like conjugate O-HTCC-SOD of the excellent efficacy resulted from its targeted intracellular ROS clearance capability and improved pharmacokinetic profiles, opening up a novel avenue for disease-modifying osteoarthritis drugs.

Efficacy and safety of levosimendan in patients with acute right heart failure: A meta-analysis.

AIMS: Right heart failure (RHF), which is caused by a variety of heart and lung diseases, has a high morbidity and mortality rate. Levosimendan is a cardiac inotropic drug and vasodilator. The effect of levosimendan on RHF remains unclear. We sought to evaluate the efficacy and safety of levosimendan in patients with acute RHF. MATERIALS AND METHODS: We systematically searched PubMed, Cochrane Library, EMBASE, and ClinicalTrials.gov to identify studies reporting the efficacy and safety of levosimendan for the treatment of RHF. KEY FINDINGS: Ten trials, including 359 participants from 6 RCTs and 4 self-controlled trials, were evaluated. In the 6 RCTs, we found that patients treated with levosimendan for 24h showed a significant increase in tricuspid annular plane systolic excursion [1.53; 95% CI (0.54, 2.53); P=0.002] and ejection fraction [3.59; 95% CI (1.21, 5.98); P=0.003] as well as a significant reduction in systolic pulmonary artery pressure [-6.15; 95% CI (-9.29, -3.02); P=0.0001] and pulmonary vascular resistance [-39.48; 95% CI (-65.59, -13.38); P=0.003], whereas changes in mean pulmonary pressure were nonsignificant. Adverse events did not significantly differ between the two groups. SIGNIFICANCE: Our study shows that levosimendan exhibits short-term efficacy for treating RHF in patients with a variety of heart and lung diseases. Additional strict multicentre RCTs with long follow-up times and large sample sizes are required to further validate the efficacy and safety of this treatment.

MiR-557 works as a tumor suppressor in human lung cancers by negatively regulating LEF1 expression.

MicroRNAs play an important role in regulating post-transcriptional gene expression in the progression of various human cancers. In this study, we investigated the role of microRNA-557 in human lung cancer cells. The molecular mechanism of microRNA-557 was also clarified in the proliferation and invasion of human lung cancer cells. Our results showed microRNA-557 levels were obviously decreased in clinical lung cancer specimens and lung cancer cell lines. Cell viability of A549 and NCI-H460 cells transfected with microRNA-557 mimics was significantly decreased than those transfected with negative control mimics. MicroRNA-557 promoted cell death of A549 and NCI-H460 but did not affect the cell apoptosis of lung cancer cells. Overexpression of microRNA-557 inhibited cell invasion of A549 and NCI-H460 cells. TargetScan analysis showed that microRNA-557 might target 3' untranslated region of lymphocyte enhancement factor 1, and the western blotting results showed that transfection of microRNA-557 mimics significantly decreased the levels of lymphocyte enhancement factor 1 in A549 and H460 cells. MicroRNA-557 might work as a tumor suppressor by negatively regulating the expression of lymphocyte enhancement factor 1 in lung cancer cells.

The values of neutrophil to lymphocyte ratio and platelet to lymphocyte ratio in predicting 30 day mortality in patients with acute pulmonary embolism.

BACKGROUND: vAcute pulmonary embolism (PE) is a life threatening disease. The treatment options depend on the severity of the disease and the mortality varies widely depending on the severity of the condition. It is important to identify patients who are at high risk of mortality. The aim of the present study was to explore the prognostic alues of neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) for 30-day mortality in patients with acute PE. METHODS: The study included 321 patients admitted to our university hospital between January 2013 and May 2015 with the diagnosis of acute PE. Multivariable risk models were developed to assess the predictive values of the NLR and PLR for 30-day mortality. Discrimination was evaluated using receiver operating characteristic (ROC) curves. RESULTS: Two hundred forty-eight patients met our selection criteria. Twenty of them died within 30 days of hospital admission. NLR was found to be an independent predicator after other confounding factors were adjusted in the model. For 1 unit of increase of NLR, the risk of 30-day mortality rose about 13  % (OR = 1.13,95 % CI: 1.04-1.23). The area under ROC for NLR is 0.79 (95 %CI: 0.703-0.880). PLR was associated with 30-day mortality in univariate analysis but the predicative ability diminished with inclusion of other predicators in multivariable model. CONCLUSIONS: NLR is readily available predicator for short-term mortality. It could be a useful indicator for identifying high risk population and guiding clinical management of acute PE.