Genes to specialized metabolites: accumulation of scopoletin, umbelliferone and their glycosides in natural populations of Arabidopsis thaliana.

BACKGROUND: Scopoletin and umbelliferone belong to coumarins, which are plant specialized metabolites with potent and wide biological activities, the accumulation of which is induced by various environmental stresses. Coumarins have been detected in various plant species, including medicinal plants and the model organism Arabidopsis thaliana. In recent years, key role of coumarins in maintaining iron (Fe) homeostasis in plants has been demonstrated, as well as their significant impact on the rhizosphere microbiome through exudates secreted into the soil environment. Several mechanisms underlying these processes require clarification. Previously, we demonstrated that Arabidopsis is an excellent model for studying genetic variation and molecular basis of coumarin accumulation in plants. RESULTS: Here, through targeted metabolic profiling and gene expression analysis, the gene-metabolite network of scopoletin and umbelliferone accumulation was examined in more detail in selected Arabidopsis accessions (Col-0, Est-1, Tsu-1) undergoing different culture conditions and characterized by variation in coumarin content. The highest accumulation of coumarins was detected in roots grown in vitro liquid culture. The expression of 10 phenylpropanoid genes (4CL1, 4CL2, 4CL3, CCoAOMT1, C3'H, HCT, F6'H1, F6'H2,CCR1 and CCR2) was assessed by qPCR in three genetic backgrounds, cultured in vitro and in soil, and in two types of tissues (leaves and roots). We not only detected the expected variability in gene expression and coumarin accumulation among Arabidopsis accessions, but also found interesting polymorphisms in the coding sequences of the selected genes through in silico analysis and resequencing. CONCLUSIONS: To the best of our knowledge, this is the first study comparing accumulation of simple coumarins and expression of phenylpropanoid-related genes in Arabidopsis accessions grown in soil and in liquid cultures. The large variations we detected in the content of coumarins and gene expression are genetically determined, but also tissue and culture dependent. It is particularly important considering that growing plants in liquid media is a widely used technology that provides a large amount of root tissue suitable for metabolomics. Research on differential accumulation of coumarins and related gene expression will be useful in future studies aimed at better understanding the physiological role of coumarins in roots and the surrounding environments.

Binding Interaction of Coumarin Derivative Daphnetin with Ovalbumin: Molecular Insights into the Complexation Process and Effects of Metal Ions and pH in the Binding and Antifibrillation Studies.

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104 M-1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions' (Cu2+ and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol-1] and ΔS° [+(181.726 ± 5.465) J mol-1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Umbelliferone protects against methylglyoxal-induced HUVECs dysfunction through suppression of apoptosis and oxidative stress.

Methylglyoxal (MGO), a cytotoxic metabolite of glycolysis, can cause endothelial cells impairment, which is tightly associated with diabetic vascular complication. Umbelliferone, a derivative of coumarin, participates in various pharmacological activities. This study aimed to determine the effectiveness of umbelliferone in MGO-induced apoptosis and oxidative stress in endothelial cells. In this study, it has been indicated that umbelliferone inhibited MGO-induced human umbilical vein endothelial cells (HUVECs) cytotoxicity, apoptosis, Bax/Bcl-2 protein ratio, the activity of cleaved-caspase-3, and mitochondrial membrane potential loss. Furthermore, we found that umbelliferone inhibited MGO-induced activation of mitogen-activated protein kinases and nuclear factor-κB signaling pathways in HUVECs. In addition, umbelliferone could suppress oxidative stress, as evidenced by decrease of reactive oxygen species and malondialdehyde (MDA) generation, and increase of superoxide dismutase and glutathione peroxidase contents. Moreover, we found that umbelliferone can activate Nrf2/HO-1 signaling. Importantly, silencing of Nrf2 signaling clearly eliminated the anti-oxidative stress of umbelliferone, whereas umbelliferone pretreatment had no effect on Nrf2 overexpressing HUVECs. Altogether, this study suggested that umbelliferone pretreatment has a protective effect on MGO-induced endothelial cell dysfunction through inhibiting apoptosis and oxidative stress.

Metabolic Syndrome Ameliorated by 4-Methylesculetin by Reducing Hepatic Lipid Accumulation.

Obesity is a chronic metabolic disease caused by an imbalance between energy intake and expenditure during a long period and is characterized by adipose tissue disfunction and hepatic steatosis. The aim of this study was to investigate the effect of 4-methylesculetin (4-ME), a coumarin derivative, upon adipose microenvironment and hepatic steatosis in mice induced by a high-fat diet (HFD), and to explore potential mechanisms of its beneficial effect on metabolic disorders. HFD-fed mice displayed visceral obesity, insulin resistance, and hepatic lipid accumulation, which was remarkably ameliorated by 4-ME treatment. Meanwhile, 4-ME ameliorated adipocyte hypertrophy, macrophage infiltration, hypoxia, and fibrosis in epididymal adipose tissue, thus improving the adipose tissue microenvironment. Furthermore, 4-ME reversed the increase in CD36, PPAR-γ, SREBP-1, and FASN, and the decrease in CPT-1A, PPAR-α, and Nrf2 translocation into the nucleus in livers of HFD mice and in FFA-incubated hepatocytes. Moreover, the beneficial effects of 4-ME upon lipid deposition and the expression of proteins related to lipid metabolism in FFA-induced LO2 cells were abolished by ML385, a specific Nrf2 inhibitor, indicating that Nrf2 is necessary for 4-ME to reduce hepatic lipid deposition. These findings suggested that 4-ME might be a potential lead compound candidate for preventing obesity and MAFLD.

[Umbelliferone improves chronic hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy].

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone.

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme.

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.

In vitro sulfonation of 7-hydroxycoumarin derivatives in liver cytosol of human and six animal species.

Sulfonation is an important high affinity elimination pathway for phenolic compounds.In this study sulfonation of 7-hydroxycoumarin and 13 its derivatives were evaluated in liver cytosols of human and six animal species. 7-hydroxycoumarin and its derivatives are strongly fluorescent, and their sulfate conjugates are nonfluorescent at excitation 405 nm and emission 460 nm. A convenient fluorescence based kinetic assay of sulfonation was established.The sulfonation rate of most of the 7-hydroxycoumarin derivatives was low in liver cytosol of human and pig, whereas it was high with most compounds in dog and intermediate in rat, mouse, rabbit, and sheep. Sulfonation of the 7-hydroxycoumarin derivatives followed Michaelis-Menten kinetics with Km values of 0.1-12 µM, Vmax of 0.005-1.7 µmol/(min * g protein) and intrinsic clearance (Vmax/Km) of 0.004-1.9 L/(min * g cytosolic protein).Fluorescence based measurement of sulfonation of 7-hydroxycoumarin derivatives provides a sensitive and convenient high-throughput assay to determine sulfonation rate in different species and tissues and can be applied to evaluate sulfonation kinetics and inhibition.

Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro.

in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.

New fluorogenic probes for neutral and alkaline ceramidases.

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

"Close-to-Release": Spontaneous Bioorthogonal Uncaging Resulting from Ring-Closing Metathesis.

Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been developed to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing metathesis (RCM). A caged molecule, tethered to a diolefinic substrate, is released via spontaneous 1,4-elimination following RCM. Using this strategy, which we term "close-to-release", we show that drugs and fluorescent probes are uncaged with fast rates, including in the presence of mammalian cells or in the periplasm of Escherichia coli. We envision that this tool may find applications in chemical biology, bioengineering and medicine.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Determine the Disposition of Esculetin-7-O-Glucuronide and 4-Methylesculetin-7-O-Glucuronide.

Esculetin (ET)-7-O-glucuronide (ET-G) and 4-methylesculetin (4-ME)-7-O-glucuronide (4-ME-G) are the main glucuronide of ET and 4-ME, respectively. The disposition mediated by efflux transporters for glucuronide has significant influence on the pharmacokinetic profile and efficacy of bioactive compounds. In the current study, transporter gene knockout mice and Caco-2 cells were used to explore the effects of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) on the disposition of ET-G and 4-ME-G. After oral or i.v. administration of ET and 4-ME, the area under the plasma concentration-time curve from time 0 to the last data point or infinity values of ET, 4-ME, and their glucuronides (ET-G and 4-ME-G) were remarkably and significantly increased in most Bcrp1-/- and Mrp2-/- mice compared with those in wild-type FVB mice (P < 0.05). These results were accompanied with a significant increase of maximum plasma concentration values (P < 0.05). In Caco-2 monolayers, the efflux and clearance rates of ET-G and 4-ME-G were markedly reduced by the BCRP inhibitor Ko143 and MRP2 inhibitor MK571 on the apical side (P < 0.05). In an intestinal perfusion study, the excretion of ET-G was significantly decreased in perfusate and increased in plasma in Bcrp1-/- mice compared with those in wild-type FVB mice (P < 0.05). The 4-ME-G concentration was also decreased in the bile in transporter gene knockout mice. ET and 4-ME showed good permeability in both Caco-2 monolayers [apparent permeability (Papp ) ≥ 0.59 × 10-5 cm/s] and duodenum (Papp ≥ 1.81). In conclusion, BCRP and MRP2 are involved in excreting ET-G and 4-ME-G. ET and 4-ME are most likely absorbed via passive diffusion in the intestines.

Enhanced intracellular uptake and stability of umbelliferone in compound mixtures from Angelica gigas in vitro.

Understanding how natural compounds work together for disease treatments can improve their clinical efficacy and therapeutic effects. To elucidate the mechanisms of synergistic biological effects in natural compound mixtures, umbelliferone (UMB, 7-hydroxycoumarin), derived from Angelica (A.) gigas, was selected as active compound with fluorescent characteristic to examine bioactivities in vitro in the presence of other compounds from Angelica gigas. Antioxidant effects of UMB in biochemical assays and cellular reactive oxygen species (ROS) levels in RAW264.7 cells were not significantly improved by addition of other compounds. However, intracellular uptake, inhibition of the efflux pump P-glycoprotein (P-gp), and physiological stability of UMB were greatly enhanced by the addition of other compounds, specifically Angelicin (ANG) and Byakangelicin (BYN). Taken together, enhanced intracellular localization and enzymatic stability in compound mixtures might lead to superior synergistic bioactivity of UMBs in compound mixtures.

Esculetin Inhibits Proliferation, Invasion, and Migration of Laryngeal Cancer In Vitro and In Vivo by Inhibiting Janus Kinas (JAK)-Signal Transducer and Activator of Transcription-3 (STAT3) Activation.

BACKGROUND Laryngeal cancer is one of the most common malignant tumors of the head and neck. Natural compounds in traditional Chinese medicine provide many valuable potential compounds for tumor chemotherapy. Esculetin, a coumarin derivative from several herbs, inhibits proliferation of many types of cancer cells, but its anticancer effect in laryngeal cancer is still not clear. MATERIAL AND METHODS We performed in vitro proliferation assay, invasion assay, and migration assay to assess the effect of esculetin against LC, and in vivo nude mouse xenograft animal model was used as well. Flow cytometry was conducted to analyze the effect of esculetin on cell cycle of LC cells, and Western blot analysis was used to assess the effect esculetin on the JAK-STAT signaling pathway. RESULTS Esculetin remarkably inhibits proliferation, migration, and invasion of LC cells, and reduces in vivo xenograft tumor growth and tumor weight in a dose-dependent manner. Our molecular mechanism study demonstrated that esculetin significantly inhibits STAT3 phosphorylation and blocks translocation of STAT3 into the nucleus, and esculetin also blocks the cell cycle in G1/S phase. CONCLUSIONS In a summary, by inhibiting the STAT3 activation, esculetin shows potential anticancer effects against the laryngeal cancer.

The Molecular and Structural Basis of O-methylation Reaction in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.

Regional Differences in Human Intestinal Drug Metabolism.

The intestines are key for the absorption of nutrients and water as well as drug metabolism, and it is well known that there are clear differences in the expression profile of drug metabolism enzymes along the intestinal tract. Yet only a few studies have thoroughly investigated regional differences in human intestinal drug metabolism. In this study, we evaluated phase I and phase II metabolism in matched human ileum and colon precision-cut intestinal slices (PCIS). To this end, human PCIS were incubated for 3 hours with testosterone and 7-hydroxycoumarin (7-HC) to examine phase I and phase II metabolism, respectively. Metabolite formation was assessed by high-performance liquid chromatography analysis. Our results demonstrated that androstenedione, 6ß-hydroxytestosterone, 2ß-hydroxytestosterone, and 7-HC sulfate were predominantly formed in the ileum, while 15α-hydroxytestosterone and 7-HC glucuronide were mainly produced in the colon. Moreover, we also observed sex differences in phase II metabolite formation, which appeared to be higher in men compared with women. Taken together, we demonstrated that phase I metabolism predominantly occurs in ileum PCIS, while phase II metabolism mostly takes place in colon PCIS. Moreover, we revealed that human PCIS can be used to study both regional and sex differences in intestinal metabolism.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4-(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.

Structure of an Insecticide Sequestering Carboxylesterase from the Disease Vector Culex quinquefasciatus: What Makes an Enzyme a Good Insecticide Sponge?

Carboxylesterase (CBE)-mediated metabolic resistance to organophosphate and carbamate insecticides is a major problem for the control of insect disease vectors, such as the mosquito. The most common mechanism involves overexpression of CBEs that bind to the insecticide with high affinity, thereby sequestering them before they can interact with their target. However, the absence of any structure for an insecticide-sequestering CBE limits our understanding of the molecular basis for this process. We present the first structure of a CBE involved in sequestration, Cqestß21, from the mosquito disease vector Culex quinquefasciatus. Lysine methylation was used to obtain the crystal structure of Cqestß21, which adopts a canonical α/ß-hydrolase fold that has high similarity to the target of organophosphate and carbamate insecticides, acetylcholinesterase. Sequence similarity networks of the insect carboxyl/cholinesterase family demonstrate that CBEs associated with metabolic insecticide resistance across many species share a level of similarity that distinguishes them from a variety of other classes. This is further emphasized by the structural similarities and differences in the binding pocket and active site residues of Cqestß21 and other insect carboxyl/cholinesterases. Stopped-flow and steady-state inhibition studies support a major role for Cqestß21 in organophosphate resistance and a minor role in carbamate resistance. Comparison with another isoform associated with insecticide resistance, Cqestß1, showed both enzymes have similar affinity to insecticides, despite 16 amino acid differences between the two proteins. This provides a molecular understanding of pesticide sequestration by insect CBEs and could facilitate the design of CBE-specific inhibitors to circumvent this resistance mechanism in the future.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Inhibition of human catechol-O-methyltransferase-mediated dopamine O-methylation by daphnetin and its Phase II metabolites.

1. Finding and developing inhibitors of catechol-O-methyltransferase (COMT) from natural products is highly recommended. Daphnetin, a naturally occurring catechol from the family thymelaeaceae, has a chemical structure similar to several potent COMT inhibitors reported previously. Here the potential of daphnetin and its Phase II metabolites as inhibitors of COMT was investigated with human liver cytosol (HLC). 2. Daphnetin and its methylated metabolite (8-O-methyldaphnetin) were found to inhibit COMT-mediated dopamine O-methylation in a dose-dependent manner. The IC50 values for daphnetin (0.51∼0.53 µM) and 8-O-methyldaphnetin (22.5∼24.3 µM) were little affected by changes in HLC concentrations. Further kinetic analysis showed the differences in inhibition type and parameters (Ki) between daphnetin (competitive, 0.37 µM) and 8-O-methyldaphnetin (noncompetitive, 25.7 µM). Other metabolites, including glucuronidated and sulfated species, showed negligible inhibition against COMT. By using in vitro-in vivo extrapolation (IV-IVE), a 24.3-fold increase in the exposure of the COMT substrates was predicted when they are co-administrated with daphnetin. 3. With high COMT-inhibiting activity, daphnetin could serve as a lead compound for the design and development of new COMT inhibitors. Also, much attention should be paid to the clinical impact of combination of daphnetin and herbal preparations containing daphnetin with the drugs primarily cleared by COMT.