In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

Application of Precision-Cut Rat Liver Slice to Study the Influence of Monocrotaline, Tussilago farfara Alkaloids on the Expression of Cytochrome P450 Enzymes

Precision-cut liver slice has been successfully used to study the mechanism of drug-induced hepatotoxicity, the prediction of liver toxicity, the discovery of early hepatic toxicity biomarker and the metabolism of drug in liver. We detected the expression of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 in precision-cut liver slice after co-cultured with monocrotaline or Tussilago farfara alkaloids to investigate the hepatotoxicity mechanism of those drugs. After co-culturing with monocrotaline or Tussilago farfara alkaloids for 6 hours, the expression of CYP3A4 in the microsome of precision-cut liver slices was detected by Western blot, and the expressions of CYP2B1 + CYP2B2 and CYP2E1 were detected by immunofluorescence. The results showed that monocrotaline induced the expression of CYP3A4 and CYP2B1 + CYP2B2, and Tussilago farfara alkaloids obviously up-regulated the expression of CYP2E1 and CYP3A4. Thus, we conclude that the up-regulation of CYP3A4, CYP2B1 + CYP2B2 and CYP2E1 may be one of the toxic mechanisms of liver injury of those drugs.

Functionalized selenium nanoparticles ameliorated acetaminophen-induced hepatotoxicity through synergistically triggering PKCδ/Nrf2 signaling pathway and inhibiting CYP 2E1

Selenium nanoparticles(SeNPs)have been demonstrated potential for use in diseases associated with oxidative stress.Functionalized SeNPs with lower toxicity and higher biocompatibility could bring better therapeutic activity and clinical application value.Herein,this work was conducted to investigate the protective effect of Pleurotus tuber-regium polysaccharide-protein complex funtionnalized SeNPs(PTR-SeNPs)against acetaminophen(APAP)-induced oxidative injure in HepG2 cells and C57BL/6J mouse liver.Further elucidation of the underlying molecular mechanism,in particular their modulation of Nrf2 signaling pathway was also performed.The results showed that PTR-SeNPs could significantly ameliorate APAP-induced oxidative injury as evidenced by a range of biochemical analysis,histopathological examination and immunoblotting study.PTR-SeNPs could hosphorylate and activate PKCδ,depress Keap1,and increase nuclear accumulation of Nrf2,resulting in upregulation of GCLC,GCLM,HO-1 and NQO-1 expression.Besides,PTR-SeNPs suppressed the biotransformation of APAP to generate intracellular ROS through CYP 2E1 inhibition,restoring the mitochondrial morphology.Furthermore,the protective effect of PTR-SeNPs against APAP induced hepatotoxicity was weakened as Nrf2 was depleted in vivo,indicating the pivotal role of Nrf2 signaling pathway in PTR-SeNPs mediated hepatoprotective efficacy.Being a potential hepatic protectant,PTR-SeNPs could serve as a new source of selenium supplement for health-promoting and biomedical applications.

A Bacterial Cytochrome P450 Enzyme Catalyzes Multistep Oxidation Reactions in Pyrroindomycin Biosynthesis

Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.

Cytochrome P450 Enzyme-Copper Phosphate Hybrid Nano-Flowers with Superior Catalytic Performances for Selective Oxidation of Sulfides

Cytochrome P450 enzyme-copper phosphate hybrid materials with flower-like shape were prepared with a simple but efficient coprecipitation method.The growth process of the hybrid flowers can be divided into three successive steps：coordination/nucleation,growth,and further ripen.The concentration of enzymes in the mother liquor exerted great influence on the morphology and surface enzyme content of the nano-composites.The catalytic performance in the reaction of selective oxidation of sulfide to sulfoxide was also investigated.The hybrid flowers exhibited superior catalytic performance：satisfied thioanisole conversion and selectivity to methyl phenyl sulfoxide （both above 97%） with H2O2 as oxidant under mild reaction conditions,excellent stability and recyclability,and wide scope of substrates.Such results indicate that the hybrid materials are potentially good candidates in the industrial enzyme catalysis.

Functional expression and regulation of eukaryotic cytochrome P450 enzymes in surrogate microbial cell factories

Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions,synthesis of diverse metabolites,detoxification of xenobiotics,and con-tribution to environmental adaptation.Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological,pharmaceutical and chemical industry applica-tions.Nevertheless,the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range.This review is focused on comprehen-sive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories,aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future.We also highlight the functional significance of the previously underrated cytochrome P450 reductases(CPRs)and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.

Regulation of the homeostasis of hepatic endoplasmic reticulum and cytochrome P450 enzymes by autophagy

The endoplasmic reticulum(ER)is an intracellular organelle consisting of a continuous network of membranes.In the liver,the ER is highly active in protein modification,lipid metabolism,and xenobiotic detoxification.Maintaining these complicated processes requires elaborate control of the ER lumen environment as well as the ER volume.Increasing evidence suggests that autophagy plays a critical role in regulating the homeostasis of hepatic ER contents and levels of cytochrome P450(CYP)enzymes via selective ER-phagy.This review will provide an overview of ER-phagy,summarizing the possible roles of recently identified ER-phagy receptor proteins in regulating the homeostasis of hepatic ER and CYP enzymes as well as outlining the various implications of ER-phagy in ER-related liver diseases.

Development of MEMS directed evolution strategy for multiplied throughput and convergent evolution of cytochrome P450 enzymes

Directed evolution(DE)inspired by natural evolution(NE)has been achieving tremendous successes in protein/enzyme engineering.However,the conventional"one-protein-for-one-task"DE cannot match the"multi-proteins-for-multi-tasks"NE in terms of screening throughput and efficiency,thus often failing to meet the fast-growing demands for biocatalysts with desired properties.In this study,we design a novel"multi-enzymes-for-multi-substrates"(MEMS)DE model and establish the proof-ofconcept by running a NE-mimicking and higher-throughput screening on the basis of"two-P450 s-against-seven-substrates"(2P×7S)in one pot.With the multiplied throughput and improved hit rate,we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes(P450 BM3 and P450 cam)in laboratory.It is anticipated that the new strategy of MEMS DE will find broader application for a larger repertoire of enzymes in the future.Furthermore,structural and substrate docking analysis of the two functionally convergent P450 variants provide important insights into how distinct P450 active-sites can reach a common catalytic goal.

Cytochrome P450s in algae:Bioactive natural product biosynthesis and light-driven bioproduction

Algae are a large group of photo synthetic organisms responsible for approximately half of the earth’s total photosynthesis.In addition to their fundamental ecological roles as oxygen producers and as the food base for almost all aquatic life,algae are also a rich source of bioactive natural products,including several clinical drugs.Cytochrome P450 enzymes(P450s) are a superfamily of biocatalysts that are extensively involved in natural product biosynthesis by mediating various types of reactions.In the post-genome era,a growing number of P450 genes have been discovered from algae,indicating their important roles in algal life-cycle.However,the functional studies of algal P450s remain limited.Benefitting from the recent technical advances in algae cultivation and genetic manipulation,the researches on P450s in algal natural product biosynthesis have been approaching to a new stage.Moreover,some photoauto trophic algae have been developed into "photo-bioreactors" for heterologous P450s to produce high-value added pharmaceuticals and chemicals in a carbon-neutral or carbon-negative manner.Here,we comprehensively review these advances of P450 studies in algae from 2000 to 2021.

Enzymatic synthesis of indigo derivatives by tuning P450 BM3 peroxygenases

Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.

Interactions of drug-metabolizing enzymes with the Chinese herb Psoraleae Fructus

Psoraleae Fructus(the dried fruits of Psoralea corylifolia), one of the most frequently used Chinese herbs in Asian countries, has a variety of biological activities. In clinical settings, Psoraleae Fructus or Psoraleae Fructus-related herbal medicines frequently have been used in combination with a number of therapeutic drugs for the treatment of various human diseases, such as leukoderma, rheumatism and dysentery. The use of Psoraleae Fructus in combination with drugs has aroused concern of the potential risks of herb-drug interactions(HDI) or herb-endobiotic interactions(HEI). This article reviews the interactions between human drug-metabolizing enzymes and the constituents of Psoraleae Fructus;the major constituents in Psoraleae Fructus, along with their chemical structures and metabolic pathways are summarized, and the inhibitory and inductive effects of the constituents in Psoraleae Fructus on human drug-metabolizing enzymes(DMEs), including target enzyme(s), its modulatory potency, and mechanisms of action are presented. Collectively, this review summarizes current knowledge of the interactions between the Chinese herb Psoraleae Fructus and therapeutic drugs in an effort to facilitate its rational use in clinical settings, and especially to avoid the potential risks of HDI or HEI through human DMEs.

A review:biosynthesis of plant-derived labdane-related diterpenoids

Plant-derived labdane-related diterpenoids(LRDs)represent a large group of terpenoids.LRDs possess either a labdane-type bicyclic core structure or more complex ring systems derived from labdane-type skeletons,such as abietane,pimarane,kaurane,etc.Due to their various pharmaceutical activities and unique properties,many of LRDs have been widely used in pharmaceutical,food and perfume industries.Biosynthesis of various LRDs has been extensively studied,leading to characterization of a large number of new biosynthetic enzymes.The biosynthetic pathways of important LRDs and the relevant enzymes(especially diterpene synthases and cytochrome P450 enzymes)were summarized in this review.

Impact of gamma-glutamyl carboxylase gene genovariation in Chinese Han population on the response of warfarin initial anticoagulant therapy

Background In recent years, it is found that the polymorphisms of genes which are involved in the pharmacokinetic and pharmacodynamic pathways play important roles in the clinical anticoagulation treatment with warfarin. The aim of the study was to investigate the impact of the genetic polymorphism of r-glutamic acid carboxylase （gamma-glutamyl carboxylase gene, GGCX） on the response of warfarin initial anticoagulant therapy. Methods Seven hundred and ninety-eight Chinese Han patients who received valve replacement surgery and orally taken warfarin in long term for anticoagulant therapy in Guangdong General Hospital from 2000 to 2008 were enrolled in the study, a polymorphic SNPs point （rs699664） of GGCX was selected, and SnaPshot was adopted to perform single nucleotide polymorphism （SNP） test, and by grouping according to genotype, GGCX average daily dose of warfarin, time for PT-INR to reach the target value and differences in the incidence of excessive coagulation between different genotypes were compared respectively. Hardy-Weinberg genetic equilibrium test was applied for population representative test. Results Within the 20 days of warfarin initial therapy, male average daily dose of warfarin （2.92 ± 1.18 mg/d） was apparently higher than that of female （2.64 ± 0.98 mg/d）, while there were no significant differences in the average time required for PT-INR to reach the target value （ 1.8） and the excessive coagulation ratio at the initial therapy stage between male and female. And there were no significant differences in the average daily dose of warfarin, time to reach the target value and excessive coagulation ratio among different GGCX genotypes. Conclusions GGCX genovariation had no significant impact on the warfarin daily dose within the 20-day initial therapy of Chinese Han Population, and for the conventional dosage program, the risk of bleeding in the GGCX mutation individuals did not increase obviously at the initial administration period.

Identification of CYP 2A6 inhibitors in an effort to mitigate the harmful effects of the phytochemical nicotine

Aim:In this study,our goal was to study the inhibition of nicotine metabolism by P4502A6,as a means for reduction in tobacco use and consequently the prevention of smoking-related cancers.Nicotine,a phytochemical,is an addictive stimulant,responsible for the tobacco-dependence in smokers.Many of the other phytochemicals in tobacco,including polycyclic aromatic hydrocarbons,N-nitrosamines,and aromatic amines,are potent systemic carcinogens.Tobacco smoking causes about one of every five deaths in the United States annually.Nicotine plasma concentration is maintained by the smokers’smoking behavior within a small range.Nicotine is metabolized by cytochrome P450s 2A6 and 2A13 to cotinine.This metabolism causes a decrease in nicotine plasma levels,which in turn leads to increased tobacco smoking,and increased exposure to the tobacco carcinogens.Methods:Using the phytochemical nicotine as a lead structure,and taking its interactions with the P4502A6 binding pocket into consideration,new pyridine derivatives were designed and synthesized as potential selective mechanism-based inhibitors for this enzyme.Results:The design and synthesis of two series of novel pyridine-based compounds,with varying substituents and substitution locations on the pyridine ring,as well as their inhibitory activities on cytochrome P4502A6 and their interactions with its active site are discussed here.Substitutions at position 3 of the pyridine ring with an imidazole or propargyl ether containing group showed the most optimal interactions with the P4502A6 active site.Conclusion:The pyridine compounds with an imidazole or propargyl ether containing substituent on position 3 were found to be promising lead compounds for further development.Hydrogen-bonding interactions were determined to be crucial for effective binding of these molecules within the P4502A6 active site.