Structure-activity relationship and inhibition of human cytochrome CYP2A6 and CYP2A13 by flavonoids.

Cigarette smoking is one of the major risk factors of various diseases including respiratory diseases and lung cancer. While the liver-specific CYP2A6 is associated with the nicotine clearance and smoking addiction, the metabolic activation of the tobacco-specific nitrosamine by lung-specific CYP2A13 can lead to lung tumorigenesis.It has been reported that inhibition of CYP2A6 and CYP2A13 enzymes by flavonoids constituents could be an aids in smoking cessation.

Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase.

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling.

Anti-proliferative effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) isolated from Vernonia cinerea on oral squamous cell carcinoma through inhibition of STAT3 and STAT2 phosphorylation.

Background: The high mortality rate of oral cancers has stimulated the search for effective herbal medicines and their pharmacological targets. Vernonia cinerea, a perennial tropical herb, is wildly used as a traditional folk medicine for treatment of intestinal diseases and various skin diseases in addition to possessing anti-cancer activity. However, the effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) as a major sesquiterpene lactone compound found in V.

Effects of Vernonia cinerea Compounds on Drug-metabolizing Cytochrome P450s in Human Liver Microsomes.

Vernonia cinerea has been widely used in traditional medicines for various diseases and shown to aid in smoking abstinence and has anticancer properties. V. cinerea bioactive compounds, including flavonoids and hirsutinolide-type sesquiterpene lactones, have shown an inhibition effect on the nicotine-metabolizing cytochrome P450 2A6 (CYP2A6) enzyme and hirsutinolides reported suppressing cancer growth.

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea.

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1-4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5-8 from V.

Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes.

The cytochrome P450 monooxygenases play a major role in insecticide detoxification and become a target for development of insecticide synergists. In this study, a collection of rhinacanthins (rhinacanthin-D, -E, -G, -N, -Q, and -H/I) purified from Rhinacanthus nasutus, in addition to previously purified rhinacanthin-B and -C, were isolated. These compounds displayed various degrees of inhibition against benzyloxyresorufin-O-debenzylation mediated by CYP6AA3 and CYP6P7 which were implicated in pyrethroid resistance in Anopheles minimus malaria vector.

Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V.

Structure–function relationships of inhibition of mosquito cytochrome P450 enzymes by flavonoids of Andrographis paniculata.

The cytochrome P450 monooxygenases are known to play a major role in pyrethroid resistance, by means of increased rate of insecticide detoxification as a result of their overexpression. Inhibition of detoxification enzymes may help disrupting insect detoxifying defense system. The Anopheles minimus CYP6AA3 and CYP6P7 have shown pyrethroid degradation activity and been implicated in pyrethroid resistance.

Distal effect of amino acid substitutions in CYP2C9 polymorphic variants causes differences in interatomic interactions against (S)-warfarin.

Cytochrome P450 2C9 (CYP2C9) is crucial in excretion of commonly prescribed drugs. However, changes in metabolic activity caused by CYP2C9 polymorphisms inevitably result in adverse drug effects. CYP2C9*2 and *3 are prevalent in Caucasian populations whereas CYP2C9*13 is remarkable in Asian populations.

Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.

  Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition.

Modeling of Anopheles minimus Mosquito NADPH-cytochrome P450 oxidoreductase (CYPOR) and mutagenesis analysis.

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A.

Inhibition against mosquito cytochrome P450 enzymes by rhinacanthin-A, -B, and -C elicits synergism on cypermethrin cytotoxicity in Spodoptera frugiperda cells.

Rhinacanthus nasutus (Acanthaceae) is a shrub reported to contain insecticidal activities. The current study was conducted to determine whether R. nasutus constituents could inhibit benzyloxyresorufin O-debenzylation (BROD) mediated by CYP6AA3 and CYP6P7.

Protective efficacy of Anopheles minimus CYP6P7 and CYP6AA3 against cytotoxicity of pyrethroid insecticides in Spodoptera frugiperda (Sf9) insect cells.

Cytochrome P450 monooxygenases (P450s) are enzymes known to metabolize a wide variety of compounds including insecticides. Their overexpression leading to enhanced insecticide detoxification could result in insecticide resistance in insects. The increased mRNA expression of two P450 genes, CYP6P7 and CYP6AA3, has been previously observed in laboratory-selected deltamethrin-resistant Anopheles minimus, a major malaria vector in Southeast Asia, suggesting their role in detoxification of pyrethroids.

An expression of an insect membrane-bound cytochrome P450 CYP6AA3 in the Escherichia coli in relation to insecticide resistance in a malarial vector.

This laboratory investigation was carried out at the Faculty of Sciences, Mahidol University, Thailand during October 2007 to May 2009. The objectives of this study include: the search for heterologous expression of the cytochrome P450 CYP6AA3 enzyme of the Anopheles minimus mosquitoes in relation to Malaria disease and to provide some information on molecular mechanism of insects' pyrethroid resistance. The polymerase chain reaction aided by the Pfu DNA polymerase and some specific generated primers were used to modify the CYP6AA3 gene.

Homology modeling of mosquito cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in substrate selectivity.

Background: Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds.

Findings: Homology models of the three An.

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties.

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7.

Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure.

Aedes aegypti mosquitoes are resistant to various insecticides, including pyrethroids, throughout Thailand. We previously reported that Ae. aegypti from Mae Wong district, Nakhon Sawan Province in north-central Thailand, were resistant to insecticides, including pyrethroids (deltamethrin and permethrin), organophosphates and carbamates, and that high levels of detoxification enzymes were present.

Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism.

The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito.

Genetic structure among Thai populations of Aedes aegypti mosquitoes.

Thirty-one field populations of Aedes aegypti (L.) were compared using isozyme starch gel electrophoresis to characterize genetic variation between populations. Ae.

Characterization of Anopheles minimus CYP6AA3 expressed in a recombinant baculovirus system.

Metabolism by cytochrome P450 monooxygenases is a major mechanism implicated in resistance of insects to insecticides, including pyrethroids. We previously isolated the cytochrome P450 CYP6AA3 from deltamethrin-selected resistant strain of Anopheles minimus mosquito, a major malaria vector in Thailand. In the present study, we further investigated the role of CYP6AA3 enzyme in deltamethrin metabolism in vitro.

NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain.

Functional expression of mosquito NADPH-cytochrome P450 reductase in Escherichia coli.

A complete NADPH-cytochrome P450 reductase (CPR) cDNA was isolated from Anopheles minimus Theobald mosquitoes by using reverse transcription-polymerase chain reaction-based methods. The complete cDNA contains 2,040 bp encoding the protein of 679 amino acids, with a calculated molecular mass of 77.3 kDa The deduced amino acid sequence had a typical feature of CPR by possessing conserved domains involved in binding of flavin mononucleotide, flavin adenine dinucleotide, and NADPH cofactors.

Biochemical studies of insecticide resistance in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand.

Biochemical analysis was performed on field caught Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) mosquitoes to determine activities of enzymes including mixed function oxidases (MFO), nonspecific esterases (alpha- and beta-), glutathione-S-transferases (GST), and insensitive acetylcholinesterase (AChE). Biochemical tests were performed on F1 generation of Ae. aegypti field caught mosquitoes, while in Ae.

Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005.

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae.

Insecticide susceptible/resistance status in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand during 2003-2005.

Susceptibility baselines and diagnostic doses of the technical grade insecticides deltamethrin, permethrin, fenitrothion, and propoxur were established based on Aedes aegypti (L.), Bora (French Polynesia), a reference susceptible strain. Field-collected Aedes mosquitoes from each part of Thailand were subjected to bioassay for their susceptibility to the diagnostic doses of each insecticide.