Microsomal Cytochrome P-450 Research Articles

Semiconducting Polymer Nanobiocatalysts for Photoactivation of Intracellular Redox Reactions

AbstractAn organic semiconducting polymer nanobiocatalyst (SPNB) composed of a semiconducting polymer core conjugated with microsomal cytochrome P450 (CYP) has been developed for photoactivation of intracellular redox. The core serves as the light‐harvesting unit to initiate photoinduced electron transfer (PET) and facilitate the regeneration of dihydronicotinamide adenine dinucleotide phosphate (NADPH), while CYP is the catalytic center for intracellular redox. Under light irradiation, the semiconducting core can efficiently catalyze the generation of NADPH with a turnover frequency (TOF) 75 times higher than the reported nanosystems, ensuring the supply of the cofactor for intracellular redox. SPNB‐mediated intracellular redox thus can be efficiently activated by light in living cells to convert the model substrate and also to trigger the bioactivation of anticancer drugs. This study provides an organic nanobiocatalytic system that allows light to remotely control intracellular redox in living systems.

Semiconducting Polymer Nanobiocatalysts for Photoactivation of Intracellular Redox Reactions.

An organic semiconducting polymer nanobiocatalyst (SPNB) composed of a semiconducting polymer core conjugated with microsomal cytochrome P450 (CYP) has been developed for photoactivation of intracellular redox. The core serves as the light-harvesting unit to initiate photoinduced electron transfer (PET) and facilitate the regeneration of dihydronicotinamide adenine dinucleotide phosphate (NADPH), while CYP is the catalytic center for intracellular redox. Under light irradiation, the semiconducting core can efficiently catalyze the generation of NADPH with a turnover frequency (TOF) 75 times higher than the reported nanosystems, ensuring the supply of the cofactor for intracellular redox. SPNB-mediated intracellular redox thus can be efficiently activated by light in living cells to convert the model substrate and also to trigger the bioactivation of anticancer drugs. This study provides an organic nanobiocatalytic system that allows light to remotely control intracellular redox in living systems.

Interaction between 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro

The compound 3,4‑dichlorophenyl‑propenoyl‑sec.‑butylamine (3,4‑DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4‑DCPB and evaluate the effects of 3,4‑DCPB on the activities of CYP450 enzymes. 3,4‑DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4‑DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4‑DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of β-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB−), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4‑hydroxy‑mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6‑hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4‑DCPB (16 μg/mL, 59 μM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB− and PB+) and CYP3A4 (PB+) were not inhibited by 3,4‑DCPB in RLMs. In conclusion, the present study showed that 3,4‑DCPB was metabolized by multiple CYP450 enzymes. 3,4‑DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4‑DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm−1 d−1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Suitable albumin concentrations for enhanced drug oxidation activities mediated by human liver microsomal cytochrome P450 2C9 and other forms predicted with unbound fractions and partition/distribution coefficients of model substrates

Albumin has reportedly enhanced cytochrome P450 (P450)-mediated drug oxidation rates in human liver microsomes. Consequently, measurements of clearances and fractions metabolized could vary depending on the experimental albumin concentrations used.In this study, the oxidation rates of diclofenac and warfarin by human liver microsomes were significantly enhanced in the presence of 0.10% (w/v) bovine serum albumin, whereas those of tolbutamide and phenytoin required 1.0% and 2.0% of albumin for significant enhancement. Values of the fractions metabolized by P450 2C9 for four substrates did not markedly change in the presence of albumin at the above-mentioned concentrations.The oxidation rates of bupropion, omeprazole, chlorzoxazone and phenacetin in human liver microsomes were reportedly enhanced by 0.5%, 1%, 2% and 2% of albumin, respectively. Analysis of reported intrinsic clearance values and suitable albumin concentrations for the currently analyzed substrates and the reported substrates revealed an inverse correlation, with warfarin as an outlier.Suitable albumin concentrations were multivariately correlated with physicochemical properties, that is, the plasma unbound fractions, octanol–water partition coefficient and acid dissociation constant (r = 0.98, p<.0001, n = 10). Therefore, multiple physicochemical properties may be determinants of suitable albumin concentrations for substrate oxidations in human liver microsomes.

High salt intake shifts the mechanisms of flow-induced dilation in the middle cerebral arteries of Sprague-Dawley rats.

The goal of the present study was to examine the effect of 1 wk of high salt (HS) intake and the role of oxidative stress in changing the mechanisms of flow-induced dilation (FID) in isolated pressurized middle cerebral arteries of male Sprague-Dawley rats ( n = 15-16 rats/group). Reduced FID in the HS group was restored by intake of the superoxide scavenger tempol (HS + tempol in vivo group). The nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and selective inhibitor of microsomal cytochrome P-450 epoxidase activity N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide significantly reduced FID in the low salt diet-fed group, whereas FID in the HS group was mediated by NO only. Cyclooxygenase-2 mRNA (but not protein) expression was decreased in the HS and HS + tempol in vivo groups. Hypoxia-inducible factor-1α and VEGF protein levels were increased in the HS group but decreased in the HS + tempol in vivo group. Assessment by direct fluorescence of middle cerebral arteries under flow revealed significantly reduced vascular NO levels and increased superoxide/reactive oxygen species levels in the HS group. These results suggest that HS intake impairs FID and changes FID mechanisms to entirely NO dependent, in contrast to the low-salt diet-fed group, where FID is NO, prostanoid, and epoxyeicosatrienoic acid dependent. These changes were accompanied by increased lipid peroxidation products in the plasma of HS diet-fed rats, increased vascular superoxide/reactive oxygen species levels, and decreased NO levels, together with increased expression of hypoxia-inducible factor-1α and VEGF. NEW & NOTEWORTHY High-salt (HS) diet changes the mechanisms of flow-induced dilation in rat middle cerebral arteries from a combination of nitric oxide-, prostanoid-, and epoxyeicosatrienoic acid-dependent mechanisms to, albeit reduced, a solely nitric oxide-dependent dilation. In vivo reactive oxygen species scavenging restores flow-induced dilation in HS diet-fed rats and ameliorates HS-induced increases in the transcription factor hypoxia-inducible factor-1α and expression of its downstream target genes.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs.

Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

AbstractStructural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b5 (cyt‐b5), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver.Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Comparisons of microsomal cytochrome P450 content and enzymatic activity towards selected model substrates and insecticides in different tissues from the migratory locust (Locusta migratoria)

Cytochrome P450 monooxygenases (P450s) are important enzymes for biotransformations of various endogenous and xenobiotic substances in various organisms. In this study, we examined microsomal P450 protein content and enzymatic activity in four major detoxification tissues dissected from fifth-instar nymphs of the migratory locust (Locusta migratoria). The highest microsomal P450 protein content was found in the gastric caeca (a part of the midgut), followed by the midgut, Malpighian tubules and fat bodies. Microsomal P450s showed the highest aromatic hydroxylation, O-dealkylation and O-dearylation activities towards six of the seven model substrates examined in the fat bodies. Although the gastric caeca showed the highest P450 protein content, the enzymatic activities towards six of the seven model substrates were the lowest in this tissue. Further, the midgut, gastric caeca and fat bodies showed significant metabolic activities towards two pyrethroid insecticides (deltamethrin and fluvalinate), but no significant activities towards the other four insecticides (malathion, chlorpyrifos, carbaryl and methoprene). These results support our conclusions: 1) total P450 protein content alone cannot be reliably used to predict its enzymatic activity, and 2) insect P450 enzymatic activity is both tissue and substrate dependent.

Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

Liver Injury by Carbon Tetrachloride Intoxication in 16 Patients Treated with Forced Ventilation to Accelerate Toxin Removal via the Lungs: A Clinical Report.

Carbon tetrachloride (CCl4) is an efficient but highly toxic solvent, used in households and commercially in the industry under regulatory surveillance to ensure safety at the working place and to protect the workers’ health. However, acute unintentional or intentional intoxications by CCl4 may rarely occur and are potentially life-threatening. In this review article, therapy options are discussed that are based on a literature review of traditional poisoning cases and the clinical experience with 16 patients with acute poisoning by CCl4. Among various therapy options, the CO2-induced hyperventilation therapy will be considered in detail as the most promising approach. This special therapy was developed because only around 1% of the intoxicating CCl4 is responsible for the liver injury after conversion to toxic radicals via microsomal cytochrome P450 2E1 whereas 99% of the solvent will leave the body unchanged by exhalation. Therefore, to enhance CCl4 elimination through the lungs, CO2 is added to the inspiration air at a flow rate of 2–3 L min−1 in order to achieve hyperventilation with a respiratory volume of 25–30 L min−1. Under this therapy, the clinical course was favorable in 15/16 patients, corresponding to 93.8%. In essence, patients with acute CCl4 intoxication should be treated by forced ventilation.

In Vitro Inhibitory Effects of Synthetic Cannabinoid EAM-2201 on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

EAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors that is widely abused as an illicit recreational drug in combination with other drugs. To evaluate the potential of EAM-2201 as a perpetrator of drug–drug interactions, the inhibitory effects of EAM-2201 on major drug-metabolizing enzymes, cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were evaluated in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry (LC-MS/MS). EAM-2201 at doses up to 50 µM negligibly inhibited the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and five UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes. EAM-2201 exhibited time-dependent inhibition of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation and CYP3A4-catalyzed midazolam 1′-hydroxylation with Ki values of 0.54 µM (kinact: 0.0633 min−1), 3.0 µM (kinact: 0.0462 min−1), 3.8 µM (kinact: 0.0264 min−1) and 4.1 µM (kinact: 0.0250 min−1), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a Ki value of 2.4 µM. Based on these in vitro results, we conclude that EAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP2C19, CYP3A4 and UGT1A3.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury.

Correction: The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

[This corrects the article on p. 755 in vol. 8, PMID: 29163152.].

Cytochrome‐P450‐Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Cytochrome‐P450‐Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

AbstractAlthough membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.

Cytochrome P450 Prefers to be in Liquid-Ordered Domains in the Endoplasmic Reticulum

High-resolution mapping of protein/lipid interactions is essential to fully understand the biological function of a membrane protein, but poses tremendous challenges to most biophysical and biochemical approaches. These challenges are more severe for membrane proteins like cytochrome P450 that contain a very large soluble domain and exhibit a completely different time scale of dynamics as compared to its transmembrane domain [Durr et al., BBA Biomemb, 1768(12), 2007; Yamamoto et al., Sci Rep, 3, 2013; Yamamoto et al., Sci Rep, 7, 2017]. In this study, we use peptide-based lipid nanodiscs to “trap” the microsomal cytochrome P450 2B4 alone or also in complex with its redox partners [Zhang et al., Angew Chem, 55(14), 2016]. We effectively utilized the unique nature of peptide-based nanodiscs, that allows for the lipid exchange among nanodiscs, in order to characterize the immediate membrane environment preferred/stabilized by cytochrome P450. We report the first evidence that CYP2B4 is able to induce the formation of “raft”-like domains in a biomimetic of the endoplasmic reticulum (ER). NMR experiments were used to identify and quantitatively determine the lipids present in the nanodiscs. In addition, biophysical experiments were supported by molecular dynamics simulations to identify a sphingomyelin binding motif in P450, which is largely conserved among microsomal P450s. The protein-induced raft formation increased the thermal stability of P450 and also dramatically altered the ligand binding kinetics. These results unveil lipid/protein dynamics which can possibly contribute to the delicate mechanism of redox catalysis in lipid membrane systems [Barnaba et al., F1000Research, 6, 2017].

In Vivo Modulation of Rat Liver Microsomal Cytochrome P450 Activity by Antimalarial, Anti-HIV, and Antituberculosis Plant Medicines.

Drug interactions are key reasons for adverse drug reactions and attrition from market. Major infectious diseases causing morbidity/mortality in Ghana are malaria, tuberculosis, and HIV/AIDS. In this study, plant medicines commonly used to treat/manage these diseases in Ghana were investigated for their potential to modulate rat cytochrome P450 enzyme activities. Fluorescence and high-performance liquid chromatography–based assays were used to assess effects of antimalarial plant medicines, Fever (FEV), Mal-TF (MAL), and Kantinka terric (KT); anti-TB medicines, Chestico (CHES), CA + ST Pains + HWNT (TF), and Kantinka herbatic (KHB); and anti-HIV/AIDS medicines, Wabco (WAB), AD + T/AD (LIV) and Kantinka BA (KBA) on rat liver microsomal cytochrome P450 enzyme activities. Effects of medicines on rat biochemical and hematological parameters were also assessed. Generally, the medicines altered microsomal CYP1A1/1A2, CYP2B1/2B2, CYP2C9, and CYP2D6 activities. Only KBA elicited an increase (80%) in CYP1A1/1A2 activity. FEV, MAL, CHES, WAB, and LIV strongly inhibited the enzyme activity. All the medicines significantly inhibited CYP2C9 (24%-80%) activity. CYP2D6 activity increased after treatment with MAL, KBA, LIV, and TF. Also, MAL, WAB, LIV, KHB, and CHES increased CYP2B1/2B2 activity, while KT decrease the activity. Generally, the medicines altered liver function in the rats. Cholesterol levels declined after KBA treatment only. White and red blood cell counts, hemoglobin and hematocrit levels were significantly reduced in KT- and KBA-treated rats. Our results suggest that use of the medicines could have implications for drug interactions and safety, particularly if the medicines are administered over prolonged periods. Further investigations are imperative to establish clinical relevance of these results.