Rabbit Cytochrome Research Articles

Correction to: Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)‑transgenic Ardisia pusilla DC

Correction to: Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)‑transgenic Ardisia pusilla DC

Expression, purification, and functional reconstitution of 19F-labeled cytochrome b5 in peptide nanodiscs for NMR studies

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h) cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital) and 2 inhibitors (alpha-naphthoflavone and ketoconazole). In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.<p> </p>

Determination of heavy metal uptake in transgenic plants harbouring the rabbit CYP450 2E1 using X-ray fluorescence analysis

Transgenic Sesbania grandiflora (L.) pers (Fabaceae) and Arabidopsis thaliana (L.) (Brassicaceae) plants harbouring the rabbit cytochrome p450 2E1 enzyme were evaluated for their ability to accumulate heavy metals, potassium (K), calcium (Ca), manganese (Mn), zinc (Zn), copper (Cu), iron (Fe), lead (Pb) and bromine (Br), using X-ray Fluorescence analysis. When grown for 15 days on heavy metal-contaminated soils, transgenic cuttings of S. grandiflora and T3 A. thaliana plants recorded higher dry and fresh weight compared with their respective controls (A. thaliana and S. grandiflora plants transformed with an empty vector). Dry weight of transgenic S. grandiflora plants (0.321 g) was seven times higher than that of the wildtype (0.049 g), and the fresh weight (4.421 g) was about 4.6 times higher. Likewise, the dry weight of CYP450 2E1 A. thaliana (0.198 g) was more than eight times higher than that seen in the control (0.024 g). Moreover, Fe, Mn, K, and Ca concentrations in transgenic plants were significantly higher than those in their corresponding controls. For instance, concentrations of accumulated K (~3000 and 2000 mg/kg dry weight in S. grandiflora and A. thaliana, respectively) were significantly higher than those recorded in their corresponding controls (2500 and 1500 mg/kg, respectively). In the same vein, translocation of all studied metals from soils cultured with transgenic plants was higher than in those cultured with the control plants. In conclusion, the obtained results show the potential in using transgenic Sesbania and Arabidopsis plants harbouring the rabbit CYP450 2E1 for phytoremediation of mixed environmental contaminants.

The role of cytochrome P450 2B6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes

Recent X-ray crystal structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.

Cytochrome-P450–Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors

It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics, and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein-protein complex. To this end, we report the experimentally measured backbone amide-(15)N CSA tensors for a membrane-bound 16.7 kDa full-length rabbit cytochrome-b5 (cytb5), in complexation with a 55.8 kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The (15)N-CSAs, determined using the (15)N CSA/(15)N-(1)H dipolar coupling transverse cross-correlated rates, for free cytb5 are compared with those for the cytb5 bound to cytP4502B4. An overall increase in backbone amide-(15)N transverse cross-correlated rates for the cytb5 residues in the cytb5-cytP450 complex is observed as compared to the free cytb5 residues. Due to fast spin-spin relaxation (T2) and subsequent broadening of the signals in the complex, we could measure amide-(15)N CSAs only for 48 residues of cytb5 as compared to 84 residues of free cytb5. We observed a change in (15)N CSA for most residues of cytb5 in the complex, as compared to free cytb5, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb5 and cationic cytP450. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions of cytb5 in the complex are -184.5, -146.8, and -146.2 ppm, respectively, with an overall average value of -165.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb5, which is similar to our previously reported values for the free cytb5.

1H, 13C and 15N resonance assignments for the full-length mammalian cytochrome b5 in a membrane environment

Microsomal cytochrome b5 plays a key role in the oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, cholesterol and steroid hormones. To better understand its functional properties in a membrane mimic environment, we carried out high-resolution solution NMR studies. Here we report resonance assignments for full-length rabbit cytochrome b5 embedded in dodecylphosphocholine micelles.

X‐ray Crystal Structure of the Cytochrome P450 2B4 Active Site Mutant F297A in Complex with Clopidogrel: Insights into Compensatory Rearrangements of the Binding Pocket

Prior X‐ray crystal structures of rabbit cytochrome P450 2B4 and human P450 2B6 have revealed the pivotal role of side‐chain rearrangement of active site residues F206 and F297 in accommodating various inhibitors or substrates. To explore further the structural basis of ligand binding, the crystal structure of 2B4 F297A was solved in complex with the drug clopidogrel. The structure demonstrated the reorientation of ligand in the active site such that the acetoxy group is oriented toward the heme, whereas the thiophene moiety now extends to the void region of A297 on the I‐helix. Interestingly, movement of the I‐helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A‐virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the F297A structure or a virtual mutant where the I‐helix was also repositioned, oriented clopidogrel preferentially with either the acetoxy or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate the substrate in a distinct orientation and has important implications for understanding the role of active site residues in functional differences among related P450 enzymes. Supported by NIH grant ES03619.

Conformational Adaptation of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 Revealed upon Binding Multiple Amlodipine Molecules

Structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine have been determined by X-ray crystallography. The presence of two drug molecules suggests clear substrate access channels in each P450. According to a previously established nomenclature, amlodipine molecules were trapped in access pathway 2f in P450 2B6 and in pathway 2a or 2f in P450 2B4. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described solvent channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B', F, F', and G' and part of helix G is substantially more open in the amlodipine complexes than in the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F', and B' and the β4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states not observed with previous P450 2B structures and provide clear evidence of the substrate access channels in two drug-metabolizing P450s. In addition, the structures exhibit the versatility that can be exploited via in silico studies with other P450 2B6 ligands as large as raloxifene and itraconazole.

Phytoremediation of Trichloroethylene And Dichlorodiphenyltrichloroethane—Polluted Water Using Transgenic Sesbania Grandiflora and Arabidopsis Thaliana Plants Harboring Rabbit Cytochrome P450 2E1

Sesbania grandiflora (L.) pers (Fabaceae) and Arabidopsis thaliana (L.) (Brassicaceae) were genetically engineered to constitutively express the rabbit cytochrome p450 2E1 enzyme aiming at increasing their activity toward trichloroethylene (TCE) and dichlorodiphenyltrichloroethane (DDT) removal. Successful generation of Sesbania and Arabidopsis transgenic plants was verified using p450 2E1 specific PCR and confirmed by western blot analysis. Gas chromatography (GC) analysis revealed that small cuttings of Sesbania and third generation (F3) Arabidopsis transgenic plants exposed to TCE and DDT in small hydroponics’ vessels accumulated more TCE and DDT compared to plants transformed with the empty vector. Furthermore, both transgenic plants were more effective in breaking down TCE and DDT with a 2-fold increase in TCE metabolism. Two independent Arabidopsis lines showed that DDT was metabolized about 4-fold higher than that detected in non transformed plants. Similarly, S. grandiflora cuttings removed 51 to 90% of the added DDT compared with only 3% removal in controls transformed with the null vector. Notably, stability of rabbit cytochrome p450 2E1 was confirmed using third generation Arabidopsis plants that displayed higher potential for the removal of two important pollutants, TCE and DDT compared with the controls.

Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions are -187.9, -166.0, and -161.1 ppm, respectively, with an overall average value of -171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb(5). However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.

Dual Ligand Complexes of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 with Amlodipine Reveal Substrate Access Channels into the Active Site

Substrate access channels have been predicted previously for several mammalian P450 enzymes based mainly on molecular dynamics simulations. We now report X‐ray crystal structures of human P450 2B6 and rabbit P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine. The presence of the two drug molecules identifies clear access pathways in each P450. Based on the access channel nomenclature, 2f and 2a channels pointing towards the membrane correlate well with amlodipine access into the active site of P450 2B4. However, amlodipine appears to enter the active site of human P450 2B6 only via channel 2f. The results also indicate that key arginine residues located on the surface and at the entrance of the substrate access channels in each of the above P450s may play a crucial role in guiding substrate entry. In addition, secondary structure elements B′, F, F′, G′ and part of helix G were substantially retracted compared with the 4‐(4‐chlorophenyl)imidazole complexes of 2B4 and 2B6. Conformational variations were predominantly localized to the above elements that surround the substrate access channels as seen previously in the open P450cam structure. To our knowledge, the structures represent the first clear evidence from X‐ray crystallography of the substrate access channels into the active site in mammalian P450s. Supported by NIH grant ES03619.

Structural Analysis of Mammalian Cytochrome P450 2B4 Covalently Bound to the Mechanism-Based Inactivatortert-Butylphenylacetylene: Insight into Partial Enzymatic Activity

A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.

Theophylline metabolism by human, rabbit and rat liver microsomes and by purified forms of cytochrome P450.

The capacity of human, rabbit and rat liver microsomes and purified isozymes of cytochrome P450 to metabolize theophylline has been assessed. In all three species the 8-hydroxylation of theophylline to 1,3-dimethyluric acid (1,3-DMU) was the major pathway. In human, control rabbit and rat liver microsomes this metabolite accounted for 59, 77 and 94%, respectively, of the total metabolites formed. In both human and control rabbit liver microsomes the N-demethylation of theophylline to 1-methylxanthine (1-MX) accounted for 20% of the total metabolites formed. N-demethylation of theophylline to 3-methylxanthine (3-MX) accounted for 21% of theophylline metabolism in human microsomes but was a minor pathway in control rabbit and rat microsomes. Acetone and phenobarbitone pretreatment markedly increased the formation of 1,3-DMU by rabbit liver microsomes. Rifampicin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration caused a slight but significant increase in this pathway. In general the N-demethylation pathways in rabbit liver microsomes were refractory to induction. In the rat, the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU were all significantly increased in Aroclor 1254, dexamethasone, phenobarbitone and 3-methylcholanthrene-treated microsomes. In reconstitution experiments the polycyclic hydrocarbon inducible rabbit cytochrome P450 Forms 4 and 6 and the constitutive Form 3b all metabolized theophylline to its three metabolites. In human liver microsomes from four subjects anti-rabbit cytochrome P450 Form 4 IgG inhibited the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU by approximately 30%. These data indicate that theophylline is metabolized by multiple forms of cytochrome P450 in human, rabbit and rat liver microsomes.

NMR-Derived Models of Amidopyrine and Its Metabolites in Complexes with Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation

To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, we studied the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T(1)) relaxation NMR. Results of STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme's active site. The model-dependent relaxation rates (R(M)) determined from T(1) relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, we used distances calculated from the relaxation rates to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyls are free to rotate between two binding modes. From these bound structures, a model of N-demethylation in which the N-linked methyl functional groups rotate between catalytic and noncatalytic positions was developed. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine.

Comparison of In Vitro Metabolism of Ticlopidine by Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4

A recent X-ray crystal structure of a rabbit cytochrome P450 2B4 (CYP2B4)-ticlopidine complex indicated that the compound could be modeled with either the thiophene or chlorophenyl group oriented toward the heme prosthetic group. Subsequent NMR relaxation and molecular docking studies suggested that orientation with the chlorophenyl ring closer to the heme was the preferred one. To evaluate the predictive value of these findings, the oxidation of ticlopidine by reconstituted CYP2B4 was studied and compared with CYP2B6, in which the thiophene portion of the molecule likely orients toward the heme. In vitro incubation of ticlopidine with both enzymes yielded the same set of metabolites: 7-hydroxyticlopidine (M1), 2-oxoticlopidine (M2), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M3), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M4), ticlopidine N-oxide (M5), and ticlopidine S-oxide dimer, a dimerization product of ticlopidine S-oxide (M6). The rates of metabolite formation deviated markedly from linearity with time, consistent with the known inactivation of CYP2B6 by ticlopidine. Fitting to a first-order equation yielded similar rate constants (k(obs)) for both enzymes. However, the amplitude (R(max)) of M1 and M6 formation was 4 to 5 times higher for CYP2B6 than CYP2B4, indicating a greater residence time of ticlopidine with its thiophene ring closer to heme in CYP2B6. In contrast, CYP2B4 formed M4 and M5 in more abundance than CYP2B6, indicating an alternate orientation. Overall, the results suggest that the preferential orientation of ticlopidine in the active site of CYP2B4 predicted by X-ray crystallography and NMR studies is unproductive and that ticlopidine likely reorients within CYP2B4 to a more productive mode.

Structures of Cytochrome P450 2B4 Complexed with the Antiplatelet Drugs Ticlopidine and Clopidogrel,

Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were determined with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes. Results of ligand docking and heme-induced NMR relaxation of drug protons showed that ticlopidine was preferentially oriented with the chlorophenyl group closest to the heme. Because of its stereocenter, clopidogrel was easier to fit in the electron density and exhibited a single orientation, which points the chlorophenyl ring toward the heme. The C(α) traces of both complexes aligned very well with each other and revealed a compact, closed structure that resembles the conformation observed in two previously determined 2B4 structures with the small molecule inhibitors 4-(4-chlorophenyl)imidazole and 1-(4-chlorophenyl)imidazole. The 2B4 active site is able to accommodate small ligands by moving only a small number of side chains, suggesting that ligand reorientation is energetically favored over protein conformational changes for binding of these similarly sized molecules. Adjusting both protein conformation and ligand orientation in the active site gives 2B4 the flexibility to bind to the widest range of molecules, while also being energetically favorable.

Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy. The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfection with pcDNA-cyp4B1 vector into C6-L, the single clone expressing cyp4B1 gene was selected (C6-CL). Prodrug for various concentrations of 4-ipo or 2-AA was treated for 72h and 96h. The cell survival rate of C6-CL was determined using MTT assay and trypan-blue dye exclusion methods. By RT-PCR analysis, fLuc and CYP4B1 expression was detected in C6-CL, but not in C6. MTT assay and trypan-blue dye exclusion showed that IC50 of C6-CL was 0.3mM and <0.01mM after 4-ipo or 2-AA treatment at 96h or 72h exposure, respectively. Cell survivals of C6-CL were more rapidly reduced after treatment with 4-ipo or 2-AA than those of C6-L cells. The cell survival rate with MTT and trypan-blue dye exclusion assay was well correlated with fLuc activity in C6-CL cells. CYP4B1-based prodrug-activating gene therapy may have the potential to treat glioma and the cytotoxic effects of CYP4B1 enzyme activated 4-ipo or 2-AA in C6, and could be clearly determined by bioluminescent activity in C6-CL.

NMR‐based Structural Model of Rabbit Cytochrome P450 2B4 with the Analgesic Amidopyrine, the Intermediate Desmethylamidopyrine, and its Product Aminoantipyrine: Implications for the Structural Basis of Sequential Metabolism

Rabbit cytochrome P450 2B4 (CYP2B4) and the analgesic amidopyrine were used as prototypes of sequential drug metabolism by mammalian cytochromes P450 (CYPs). CYP2B4 metabolizes amidopyrine by two N‐demethylation steps to form the product didesmethylamidopyrine. Despite numerous studies of amidopyrine with this enzyme, little is known about the structural basis of sequential metabolism. In order to narrow our gaps in understanding, NMR studies were undertaken to build a structural model of binding of amidopyrine, the singly demethylated intermediate, and its product to CYP2B4. Heme‐induced relaxation of ligand protons was used to position the ligands in the active site of CYP2B4. Saturation transfer and NOE experiments were used to probe the protein and aqueous environments of the ligands, respectively. This information was combined and then used as constraints for a molecular dynamics simulation of ligands bound to CYP2B4. Implications of the model will be discussed in the context of sequential metabolism by CYPs as a whole and how this technology can be applied to other CYPs. (Supported by NIH grant ES003619).

Cooperativity of cytochrome P450 1A2: Interactions of 1,4-phenylene diisocyanide and 1-isopropoxy-4-nitrobenzene

Homotropic cooperativity of 1-alkoxy-4-nitrobenzene substrates and also their heterotropic cooperative binding interactions with the iron ligand 1,4-phenylene diisocyanide (Ph(NC)2) had been demonstrated previously with rabbit cytochrome P450 (P450) 1A2 [G.P. Miller, F.P. Guengerich, Biochemistry 40 (2001) 7262–7272]. Multiphasic kinetics were observed for the binding of Ph(NC)2 to both ferric and ferrous P450 1A2, including relatively slow steps. Ph(NC)2 induced an apparently rapid change in the circular dichroism spectrum, consistent with a structural change, but had no effect on tryptophan fluorescence. Ph(NC)2 binds the P450 iron in both the ferric and ferrous forms; ferric P450 1A2 was reduced rapidly in the absence of added ligands, and the rate was attenuated when Ph(NC)2 was bound. No oxidation products of Ph(NC)2 were detected. Docking studies with a rabbit P450 1A2 homology model based on the published structure of a human P450 1A2·α-naphthoflavone (αNF) complex indicated adequate room for a complex with either two 1-isopropoxy-4-nitrobenzene molecules or a combination of one 1-isopropoxy-4-nitrobenzene and one Ph(NC)2; in the case of αNF no space for an extra ligand was available. The patterns of homotropic cooperativity seen with 1-alkoxy-4-nitrobenzenes (biphasic plots of v vs. S) differ from those seen with polycyclic hydrocarbons (positive cooperativity), suggesting that only with the latter does the ligand interaction produce improved catalysis. Consistent with this view, Ph(NC)2 inhibited the oxidation of 1-isopropoxy-4-nitrobenzene and other substrates.