The development of isoform-specific probe(s) for a target enzyme with multiple homologs is always challenging. Herein, a practical strategy was used to design and develop an isoform-specific probe for CYP1A1, a key cytochrome P450 isoenzyme involved in xenobiotic metabolism and bioactivation. On the basis of the subtle differences in 3D structure and substrate preference between CYP1A1 and its homolog CYP1A2, we proposed that it was possible to design a CYP1A1-specific probe via local modification of the reaction site on known CYP1A substrates. To validate this hypothesis, 4-hydroxy-1,8-naphthalimide (HN) was selected as the basic fluorophore due to its excellent optical properties, while a series of O-alkylated HN derivatives were prepared to evaluate their specificity towards CYP1A1. Our results revealed that the introduction of a chloroethyl to HN could get the best isoform selectivity towards CYP1A1 over other CYPs including CYP1A2. The newly developed probe NBCeN exhibited excellent specificity, high sensitivity, and a ratiometric fluorescence response following CYP1A1-catalyzed O-dechloroethylation. NBCeN was successfully used to real-time monitor the activity of CYP1A1 in complex biological samples and to rapidly screen CYP1A1 modulators in living systems. NBCeN could also be used for two-photon imaging of intracellular CYP1A1 in living cells and tissues with high ratiometric imaging resolution and deep tissue penetration. All these findings demonstrated that local modification of non-specific substrates was a practical strategy to develop an isoform-specific probe for a target isoenzyme, while NBCeN could serve as a specific imaging tool to explore the biological functions of CYP1A1 in complex biological systems.

•GA derivatives were designed, synthesized and evaluated for their inhibitory activities against hCE1 and hCE2.•Compound 15 was discovered as a novel and highly selective inhibitor against hCE2 (IC50 0.02 μM).•Molecular docking revealed the essential structural features of compound 15 as potent and selective hCE2 inhibitor.Human carboxylesterase 2 (hCE2), one of the major carboxylesterases in the human intestine and various tumour tissues, plays important roles in the oral bioavailability and treatment outcomes of ester- or amide-containing drugs or prodrugs, such as anticancer agents CPT-11 (irinotecan) and LY2334737 (gemcitabine). In this study, 18β-glycyrrhetinic acid (GA), the most abundant pentacyclic triterpenoid from natural source, was selected as a reference compound for the development of potent and specific inhibitors against hCE2. Simple semi-synthetic modulation on GA was performed to obtain a series of GA derivatives. Structure-activity relationship analysis brought novel insights into the structure modification of GA. Converting the 11-oxo-12-ene of GA to 12-diene moiety, and C-3 hydroxyl and C-30 carboxyl group to 3-O-β-carboxypropionyl and ethyl ester respectively, led to a significant enhancement of the inhibitory effect on hCE2 and the selectivity over hCE1. These exciting findings inspired us to design and synthesize the more potent compound 15 (IC50 0.02 μM) as a novel and highly selective inhibitor against hCE2, which was 3463-fold more potent than the parent compound GA and demonstrated excellent selectivity (>1000-fold over hCE1). The molecular docking study of compound 15 and the active site of hCE1 and hCE2 demonstrated that the potent and selective inhibition of compound 15 toward hCE2 could partially be attributed to its relatively stronger interactions with hCE2 than with hCE1.

•A new fluorescent probe for the selective detection of human catechol-O-methyltransferase (COMT) was developed.•The probe exhibited excellent selectivity and sensitivity towards COMT.•The probe could be used to monitor the real activity of COMT in living cells.Catechol-O-methyltransferase (COMT), one of the most important phase II drug metabolizing enzymes, plays important roles in the metabolism of endogenous and xenobiotic catechols. In this study, a highly selective fluorescent probe for sensing activities of catechol-O-methyltransferase in complex biological samples was discovered and well characterized. Under physiological conditions, COMT selectively catalyzes the conversion of the probe (7,8-dihydroxy-4-methylcoumarin, DHMC) to 7-hydroxy-8-methoxy-4-methylcoumarin (HMMC), which brings a strong turn-on fluorescence signal at 520 nm. The probe substrate has been used for monitoring the real activities of COMT in complex biological samples, as well as for rapid screening of potential COMT inhibitors which are useful in the treatment of Parkinson's diseases Furthermore, the probe has been successfully used to monitor endogenous COMT in living cells for the first time, and the results demonstrate that DHMC is cell membrane permeable and low toxic to the cells. All these features of DHMC suggested that this probe holds great promise for COMT-related regulation and inhibition assays in drug discovery, as well as for further investigation on the biological functions of COMT in living cells.A highly selective fluorescent probe for catechol-O-methyltransferase.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure–selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

Baicalein (BA), a natural flavonoid compound, possesses many desirable pharmacological activities. However, poor solubility and extensive metabolism by human UDP-glucuronosyltransferases (UGTs) strongly restrict the clinical applications of BA. We previously reported that two C-8 Mannich base derivatives of BA (BA-a and BA-j) displayed enhanced solubility and anti-cyclin dependent kinase 1 activity, yet the metabolic stabilities of these compounds remained unknown. This study aimed to evaluate the in vitro glucuronidation stability of these BA derivatives and to explore the key factors affecting the UGT-mediated biotransformation. The results showed that the glucuronidation stabilities of these BA derivatives were much higher than BA. BA-a exhibited 12-fold and BA-j exhibited 5-fold improved stability in human liver S9, while in human intestine S9, BA-a and BA-j exhibited 42-fold and 33-fold improved stability, respectively. Further investigations found that the major glucuronidation site(s) were changed from 7-OH and 6-OH in BA to 6-OH in the BA derivatives. Also, both the involved enzymes and their catalytic efficacy in 6-O-glucuronidation of BA derivatives were much lower than that of BA. The formation of an intramolecular hydrogen bond between the C-8 Mannich base substituents and C-7 phenolic groups played a predominant role in these glucuronidation changes. The calculated bond dissociation energy (BDE) of each phenolic group in BA and its derivatives agreed well with their glucuronidation activities. All these findings bring new insights into the structure–glucuronidation relationship and provide a practical strategy for the structural modification to improve the glucuronidation stability of drug candidates, especially for those phenolic compounds.

•A ratiometric fluorescent probe has been developed for UGT1A1 for the first time.•The probe triggers remarkable changes in the color and fluorescence spectrum.•The probe can monitor the enzyme activity of UGT1A1 in complex biological systems.•The probe can also be used to image endogenous UGT1A1 in living cells.This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated.

•A highly selective ratiometric fluorescent probe of hCE1 was synthesized and well-characterized for the first time.•Isoform specific hydrolysis mediated by hCE1 leads to a large emission shift with a desirable red-shifted.•The fluorescence intensity ratio (488 nm/368 nm) exhibited a good linearity increasing with both protein concentration and time.•The probe was successfully applied in measuring hCE1 activity and screening the inhibitors of hCE1 in a heterogeneous mixture containing multi-enzymes.•The probe was cell membrane permeable and can be used to image the hCE1 activity in living cells.A new ratiometric fluorescent probe derived from 2-(2-hydroxy-3-methoxyphenyl) benzothiazole (HMBT) has been developed for selective monitoring of human carboxylesterase 1 (hCE1). The probe is designed by introducing benzoyl moiety to HMBT. The prepared latent spectroscopic probe 1 displays satisfying stability under physiological pH conditions with very low background signal. Both the reaction phynotyping and chemical inhibition assays demonstrated that hCE1 mediated the specific cleavage of the carboxylic ester bond of probe 1 in human biological samples. The release of HMBT leads to a remarkable red-shifted emission in fluorescence spectrum (120 nm large emission shift). Furthermore, human cell-based assays show that probe 1 is cell membrane permeable, and it can be used for bioassay and cellular imaging of hCE1 activity in HepG2 cells. These findings lead to the development of a simple and sensitive fluorescent method for measurement of hCE1 activity in vitro or in living cells, in the presence of additional enzymes or endogenous compounds.

Diethylstilbestrol (DES), a synthetic estrogen, is famous for its carcinogenic effects. Human exposure to this compound can occur frequently through dietary ingestion and medical treatment. Glucuronidation has been demonstrated to be a predominant metabolic pathway for DES in human. Therefore, glucuronidation metabolism may have a significant impact on its toxicities, and it is essential to clarify this metabolic pathway. Accordingly, this in vitro study is designed to characterize the UGTs involved in DES glucuronidation and, furthermore, to identify the roles of individual isoforms in the reaction in liver and intestine. Human liver microsomes (HLM) displayed much higher potential for DES glucuronidation than human intestinal microsomes (HIM). The intrinsic clearances in HLM and HIM were demonstrated to be 459 and 14 μL/min/mg protein, respectively. Assays with recombinant UGTs demonstrated that UGT1A1, -1A3, -1A8, and -2B7 could catalyze DES glucuronidation, among which UGT2B7 showed the highest affinity. Chemical inhibitors of UGT2B7 and UGT1A1/1A3 both displayed similar inhibition against the reaction in UGT2B7 and HLM. In addition, DES glucuronidation in individual HLM exhibited a large individual variability and strongly correlated to UGT2B7 activity. All evidence indicates that UGT2B7 may act as a major enzyme responsible for DES glucuronidation in human liver. For HIM, both UGT2B7 inhibitor and UGT1A1/1A3/1A8 inhibitor exerted moderate inhibition. It is suggested that although UGT2B7 contributes to DES glucuronidation in intestine, other UGTs may contribute equally. In summary, this study characterizes human UGTs involved in DES glucuronidation in human liver and intestine, which may be helpful for further study about DES-related toxicities.

In silico approaches have become an alternative method to study O-glycosylation. In this paper, we developed a linear interpretable model for O-glycosylation prediction based on an unbalanced dataset, analyzing the underlying biological knowledge of glycosylation. A training set of 4446 sites involving 468 positive sites and 3978 negative sites was developed during this research. The sites were encoded using the amino acid index (AAindex), and the forward stepwise procedure utilized for feature selection. The linear discriminant analysis with an equal a priori probability (PP-LDA) was employed to develop the interpretable model. Performance of the model was verified using both the internal leave-one-out cross-validation and external validation methods. Two non-linear algorithms, the supervised support vector machine and the unsupervised self-organizing competitive neural network, were used as comparisons. The PP-LDA model exhibited improved classification results with accuracy of 82.1 % for cross-validations and 80.3 % for external prediction. Further analysis of this linear model indicated that the properties at position R1 and the properties relative to hydrophobicity contributed more to the glycosylation prediction. However, the alpha and turn propensities at the C-terminal, together with physicochemical properties at the N-terminal, are also relative to the glycosylation activity. This model is not only capable of predicting the possibility of glycosylation using an unbalanced dataset, but is also helpful to understand the underlying biological mechanisms of glycosylation. Considering the publicly accessibility of our prediction model, a downloadable program is provided in our supply materials.

Taxus mairei is a critically endangered and commercially important cultured medicinal gymnosperm in China and forms an important medicinal resource, but the research of its genome is absent. In this study, we constructed a T. mairei fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of one million clones with an average insert size of about 39 kb, amounting to 3.9 genome equivalents. Fosmid stability assays indicate that T. mairei DNA was stable during propagation in the fosmid system. End sequencing of both 5′ and 3′ ends of 968 individual clones generated 1,923 sequences after trimming, with an average sequence length of 839 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 560 (29.1%) significant hits (E < e−5). Repetitive sequences analysis revealed that 20.8% of end sequences are repetitive elements, which were composed of retroelements, DNA transposons, satellites, simple repeats, and low complexity sequences. The distribution pattern of various repeat types was found to be more similar to the gymnosperm Pinus and Picea than to the monocot and dicot. The satellites of T. mairei were significantly longer than those of P. taeda and P. glauca. The tetra-nucleotide repeats of T. mairei were much longer than those of P. glauca and P. taeda. The fosmid library and the fosmid end sequences, for the first time, will serve as a useful resource for large-scale genome sequencing, physical mapping, SSR marker development and positional cloning, and provide a better understanding of the Taxus genome.

Seven pairs of epimers and one pair of isomeric metabolites of taxanes, each pair of which have similar structures but different retention behaviors, together with additional 13 taxanes with different substitutions were chosen to investigate the quantitative structure-retention relationship (QSRR) of taxanes in ultra fast liquid chromatography (UFLC). Monte Carlo variable selection (MCVS) method was adopted to choose descriptors. The selected four descriptors were used to build QSRR model with multi-linear regression (MLR) and artificial neural network (ANN) modeling techniques. Both linear and nonlinear models show good predictive ability, of which ANN model was better with the determination coefficient R2 for training, validation and test set being 0.9892, 0.9747 and 0.9840, respectively. The results of 100 times’ leave-12-out cross validation showed the robustness of this model. All the isomers can be correctly differentiated by this model. According to the selected descriptors, the three dimensional structural information was critical for recognition of epimers. Hydrophobic interaction was the uppermost factor for retention in UFLC. Molecules’ polarizability and polarity properties were also closely correlated with retention behaviors. This QSRR model will be useful for separation and identification of taxanes including epimers and metabolites from botanical or biological samples.

The distinct inhibitory effects against CYP2D6 enzyme of the stereoisomers quinidine and quinine were investigated in this work by employing various methods, including the comparative molecular field analysis (CoMFA), the comparative molecular similarity indices analysis (CoMSIA), the molecular electrostatic potential (MEP) analysis and the docking method. Several 3D-QSAR models with proper reliability were well established, with a CoMFA model with steric and electrostatic fields exhibiting 0.67, 0.99 and 0.88 of q2, r2 and rpred2, respectively, a CoMSIA model with steric, electrostatic and H-bond acceptor fields displaying 0.72, 0.97 and 0.84 of q2, r2 and rpred2, respectively. These models and related docking results reveal that quinidine binds to CYP2D6 in an inverse orientation as compared with quinine. Moreover, quinidine blocks the entrance of the active pocket of CYP2D6 more closely than quinine does, which explains well why the inhibitory activity of quinidine is of 2 magnitudes larger than quinine. This investigation provides a better understanding of the stereoisometric effects on the bioactivities of the chiral isomers quinidine and quinine interacting with CYP2D6.The different modes of the stereoisomers bound to CYP2D6 are resulted from the different molecular electrostatic potential surface.

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.

A simple and sensitive method for determination of the O-demethylation activity of rat, dog, minipig, and human liver micrsomes toward paeonol using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized O-demethylated metabolite of paeonol (2,4-dihydroxyacetophenone, DHA) as a standard for method validation. Validation was done with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm), with phase of acetonitrile–water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for DHA was linear from 0.5–100 μM with r2 = 0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in paeonol O-demethylation (709.7 pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6 pmol/min/mg protein), HLMs (569.3 pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3 pmol/min/mg protein). The developed method was appropriated for rapid screening paeonol O-demethylation activity in liver microsomes from different species.

The precise delimitation of Taxaceae and Cephalotaxaceae is not totally resolved. Some contradicting taxonomic proposals have been published, which demonstrates the difficulties in establishing a natural classification of the families and especially in proposing a relevant treatment within the genera Taxus and Cephalotaxus. The aims of this study are to contribute to the phylogeny and specific delineation of the two conifer families on the basis of molecular data. A cladistic analysis of the sequences of five chloroplast (matK, rbcL, trnL, trnL-trnF spacer, and psbA-trnH spacer) and one nuclear (ITS) molecular markers was carried out, both individually and in combination, by distance, parsimony, likelihood, and Bayesian methods. The results confirm that the two families are monophyletic. In the genus Taxus, T. floridana is the first-branching taxon; T. brevifolia and T. globosa cluster together and are sister to T. baccata; the endemic T. yunnanensis clusters with T. wallichiana in subclade B and is only distantly related with the other four Taxus species in China (subclade A); T. fuana is closer to T. baccata than to other Taxus species. Torreya jackii and A. formosana are the first-branching species within Torreya and Amentotaxus, respectively. C. koreana and C. wilsoniana could be treated as two varieties of C. harringtonia. The ancestral distribution area of Taxaceae and Cephalotaxaceae is restricted either to Southwest China or Southeast China by DIVA analysis. The relaxed molecular clock indicates that the deepest divergences in Taxus go back to the late-Cretaceous. psbA-trnH, rbcL third codon position, and matK first codon position contributed most to the separation of taxa in Discriminant function analysis. Our results confirm, on a basis of multiple molecular markers and a complete sampling of basic species, the suggested monophyly of Taxaceae and Cephalotaxaceae and propose interspecific relationships within each group, with profound nomenclatural and taxonomic implications. Combination of partitioned Bayesian analysis and likelihood-based methods produced a more robust phylogenetic hypothesis for the two studied families.

A rapid and specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3′-azido-3′-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm), with phase of acetonitrile–water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5–500 μM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 °C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.

Cytochrome P450 (CYP, P450) 2C9 is one of three human microsomal CYPs in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The enhancement of recombinant CYP2C9 expression of a transformed E. coli strain is essential for the development of an efficient large-scale bioprocess. The recombinant CYP2C9 production is influenced by various factors, especially the medium components. The aim of this study is to optimize the culture medium of the recombinant E. coli, to determine the influence order of 11 factors, and to improve the yield of heterologously expressed CYP2C9. Plackett–Burman (PB) design, a statistical methodology, was used to screen 11 nutrients and medium components for the production of human CYP2C9 from E. coli BL21(DE3)pLysS harboring plasmid pCW2C9dH in aerobic shaking flask cultures. The experimental data were subject to statistical analysis for calculating the regression coefficients. The matrix of PB design was also used to construct the series that is needed for grey relational analysis (GRA), an approach totally different from traditional statistical analysis used to calculate the relational grade between two sequence data, to determine the influence priority of 11 parameters. Influence priority: glycerol > δ-ALA > IPTG = ampicillin > chloramphenicol = inoculum density > peptone > thiamine > trace elements > NH4Cl > MgSO4. The coefficient for the individual factor estimated from PB design agrees the conclusion of GRA. This is the first report that combined two powerful analysis methods for the enhanced production of recombinant protein.

In order to determine Km values of substrates for CYP3A4-mediated metabolism, an in silico model has been developed in the present work. Using electrotopological state (E-state) indices, together with Bayesian-regularized neural network (BRNN), we have described an in silico method to model log (1/Km) values of various substrates. The relative importance of the E-state indices is analyzed by principal component analysis. By using an additional external test set, which is independent of the training set, the robustness and predictivity of the model are also validated.Using electrotopological state (E-state) indices, together with Bayesian-regularized artificial neural network, we describe an in silico approach for modeling the CYP3A4 enzyme kinetics.

Cytochrome P450 3A4 (CYP3A4), a major member of cytochrome P450 isoenzymes, metabolizes the majority of steroids in 6β-position. For the purpose of determining requisite structural features of a series of structurally related steroids for CYP3A4-mediated metabolism, three-dimensional pharmacophore modeling as well as electrotopological state map were conducted for 15 steroids. Though prior studies speculated that the chemical reactivity of the allylic 6β-position might have a greater influence on CYP3A4 selective 6-hydroxylation than steric constraints in the enzyme, our results reveal that for CYP3A4 steroidal substrates, it is not the chemical reactivity of atoms at 6β-site, but the pharmacophoric features, i.e. the two hydrophobic rings together with two H-bond donors, that act as the key factors responsible for determining the CYP3A4 selective 6-hydroxylation of steroids.

•Efficient preparation of esculentoside B was achieved by biotransformation.•β-d-glucosidase from snailase was used to perform such biotransformation.•Response surface methodology was used to optimize the reaction conditions.•Kinetic studies determined the catalytic efficiency in this preparation process.Esculentoside B (EsB, also named phytolaccagenin 3-O-β-d-xylopyranoside), a pentacyclic triterpene isolated from herbal medicine Radix phytolaccae, has been found to possess multiple pharmacological activities. Nonetheless, the low content in nature and the difficulties in the total synthesis of EsB strongly limit its extensive investigations and further development as a drug candidate. This study aims to provide a practical method for highly efficient preparation of EsB using esculentoside A (EsA, phytolaccagenin 3-O-β-d-glucopyranosyl (1 → 4)-β-d-xylopyranoside) as the starting material. β-d-glucosidase from snailase was used to catalyze the formation of EsB, and the product was then purified and fully characterized by both HRMS and NMR. To prepare EsB in a more cost-effective way, response surface methodology (RSM) was used to explore the potential effects of the reaction conditions (such as reaction temperature, pH, enzyme load, and reaction time) on the conversion rates of EsA. The highest EsB yield of 0.66 mg/ml was obtained experimentally under optimized conditions as follows: temperature 48.28 °C, pH 6.4, enzyme load 4.43%, and reaction time 2.73 h. This result agreed well with the predicted yield of 0.68 mg/ml by RSM. The enzymatic kinetics of this biotransformation was characterized at the optimum pH and temperature. The S50 value was evaluated as 167.4 μM, while the Vmax value was 345.6 nmol/min/mg. In summary, this study provided a mild and practical method for the highly efficient preparation of EsB from EsA, which held great promise for large scale production of EsB.Download full-size image

A strain of Arthrobacter sp. DL001 with high transglycosylation activity was successfully isolated from the Yellow Sea of China. To purify the extracellular enzyme responsible for transglycosylation, a four-step protocol was adopted and the enzyme with electrophoretical purity was obtained. The purified enzyme has a molecular mass of 210 kDa and displays a narrow hydrolysis specificity towards α-1,4-glucosidic bond. Its hydrolytic activity was identified as decreasing in the order of maltotriose > panose > maltose. Only 3.61% maltose activity occurs when p-nitrophenyl α-d-glycopyranoside serves as a substrate, suggesting that this enzyme belongs to the type II α-glucosidase. In addition, the enzyme was able to transfer glucosyl groups from the donors containing α-1,4-glucosidic bond specific to glucosides, xylosides and alkyl alcohols in α-1,4- or α-1,6-manners. A decreased order of activity was observed when maltose, maltotriose, panose, β-cyclodextrin and soluble starch served as glycosyl donors, respectively. When maltose was utilized as a donor and a series of p-nitrophenyl-glycosides as acceptors, the glucosidase was capable of transferring glucosyl groups to p-nitrophenyl-glucosides and p-nitrophenyl-xylosides in α-1,4- or α-1,6-manners. The yields of p-nitrophenyl-oligosaccharides could reach 42–60% in 2 h. When a series of alkyl alcohols were utilized as acceptors, the enzyme exhibited its transglycosylation activities not only to the primary alcohols but also to the secondary alcohols with carbon chain length 1–4. Therefore, all the results indicated that the purified α-glucosidase present a useful tool for the biosynthesis of oligosaccharides and alkyl glucosides.Graphical abstractDownload full-size imageHighlights► A strain of Arthrobacter sp. DL001 with high transglycosylation activity was successfully isolated from China Yellow Sea. ► The enzyme with transglycosylation activity from Arthrobacter sp. DL001 was purified by a four-step process and identified as type II glucosidase according to their substrate specificity. ► The transglycosylation study of the enzyme indicated that it was able to transfer glucosyl groups from donors containing α-1,4-glucosidic bond specific to glucosides, xylosides and alkyl alcohols in α-1,4- or α-1,6-manners.

A broad specificity β-d-glycosidase, designated G I, was purified to homogeneity from the viscera of the China white jade snail (Achatina fulica). The enzyme was a monomeric protein with molecular weight of 115 kDa. G I has broad glycone specificity towards p-nitrophenyl derivatives of β-d-glucose, β-d-fucose, β-d-galactose, α-d-glucose and some disaccharides. The optimum pH and temperature are 5.5 and 55 °C, respectively. It was stable over a wide pH range (5–10 at 30 °C for 24 h) and against a relatively high temperature (50 °C for 4 h). Moreover, it was also stable and active in the presence of various alcohols. With pNPGlu, pNPFuc and cellobiose as donor, G I showed high transglycosylation activity. Six transglycosylation products were isolated from the reaction mixture containing 20% alcohol as glycoside acceptor using a preparative thin layer chromatography (preparative TLC) and identified as alkyl-glucosides and alkyl-fucosides by mass spectrometry (MS) analysis. Combining the high alcohol tolerance, moderate temperature and pH stability and alkyl glycosides production efficiency through transglycosylation, G I can be considered to be a promising candidate for the production of various alkyl glycosides.

A β-d-glycosidase (G I) from the China white jade snail showed non-Michaelis–Menten mode in catalyzing the reaction using pNPGlu and pNPFuc as the substrate and monitoring the released pNP. We determined quantitatively both the transglycosidic and hydrolytic products of pNPGlu and pNPFuc solvolysis for the detailed kinetic analysis on G I-catalyzed hydrolysis and transglycosylation reaction. The inhibition kinetic studies using deoxynojirimycin (DNJ) and butanol as inhibitors were preceded. DNJ only inhibited competitively the hydrolysis of cellobiose and pNPGlu while “activated” the transglycosylation of pNPGlu and pNPFuc. This was evident from the increased Vmaxtr value with no change of the apparent Kmtr. In contrast, butanol exhibited a competitive inhibition to the transglycosylation reaction and non-competitive inhibition to the hydrolysis. The results indicated that the non-Michaelis–Menten kinetic behavior was caused by the co-occurrence of substrate transglycosylation reaction. This study provided a simple method to increase the transglycosylation yield by using DNJ to inhibit hydrolysis.

Boc5, the first nonpeptidic agonist of Glucagon-like peptide-1 receptor, has been recognized as a potential candidate for treatment of diabetes. However, the metabolic behaviors of this novel molecule in both human and experimental animals remain unclear. This study aimed to explore the metabolic behaviors of Boc5 in biological preparations from human, pig and rat. Boc5 was found to be very stable in liver microsomes of human, pig and rat, but it can be degraded to two metabolites in plasma from all three species, via the successive hydrolysis of the C-22 esters. Chemical inhibition studies using selective esterase inhibitors and assays with purified enzymes suggested that Boc5 hydrolysis in human was totally mediated by human serum albumin (HSA) rather than esterases. ESI-TOF-MS/MS analysis revealed that Lys525 of HSA could be modified by treatment with Boc5, strongly suggesting the pseudo-esterase activity of albumin. Studies on species differences in this albumin-mediated metabolism showed large species differences in degradation rate of Boc5, the half lives of Boc5 in plasma from three various species varied from 23.5 h to 83.1 h, but they were much closer to the half lives of Boc5 in corresponding serum albumins, implying the predominant role of serum albumin in plasma metabolism of Boc5. Additionally, the effects of various ligands including fatty acids and several drugs with unambiguous binding sites on HSA, on the pseudo-esterase activity of HSA, were also investigated using both experimental and molecular modelling studies. These results showed that the binding of various ligands to HSA could significantly affect the pseudo-esterase activity of HSA towards Boc5, due to the ligand-induced conformation changes of HSA.Download high-res image (73KB)Download full-size image

•E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES.•E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES.•Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the activity of UGT1A4.This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0–6.25 μM), Km values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while Vmax values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate Vmax from 0.016 to 0.81 nmol/min/mg, while lifting Km in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with KA, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4.Download high-res image (201KB)Download full-size image