A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function.

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.

Amino acid residues critical for differential inhibition of CYP2B4, CYP2B5, and CYP2B1 by phenylimidazoles.

The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.

Differential activities of CYP1A isozymes in hepatic and intestinal microsomes of control and 3-methylcholanthrene-induced rats.

Differences in expression of CYP1A isoforms (CYP1A1 and CYP1A2) in liver and small intestine of male Wistar rats and their inducibility by 3-methylcholanthrene as well as the effect of different CYP1A1/1A2 expression on caffeine metabolism were investigated. In rat liver, CYP1A2 is the predominant isoform and CYP1A1 protein expression in liver is significantly increased after treatment by 3-methylcholanthrene. In contrast, only CYP1A1 was detected in control and 3-methylcholanthrene induced small intestine microsomes. Treatment with 3-methylcholanthrene (40 mg/kg intraperitoneally daily during 1, 2, 3 or 4 days) demonstrated that liver CYP1A1 is more sensitive for the induction effects than CYP1A2 and also that significant induction of CYP1A1 in rat small intestine only occurred after 3 to 4 days pretreatment. Caffeine metabolism and inhibition studies by furafylline, CYP1A1 antiserum and ketoconazole revealed that the differences in the expression of CYP1A1 and CYP1A2 in the two tissues led to significant changes in the contribution of the various isoenzymes involved in the biotransformation of caffeine. Whereas in liver paraxanthine formation was almost exclusively catalyzed by CYP1A2, in rat proximal intestine it was formed by CYP1A1. In addition, other CYP enzymes (most probably CYP3A) play a significant role in theobromine and theophylline formation from caffeine in rat intestine. Overall, this study shows different expression and inducibility of CYP1A1/1A2 by 3-methylcholanthrene in rat liver and small intestine. Furthermore in rat intestine cytochrome P450 isozymes such as CYP1A1 and CYP3A replace CYP1A2 in the caffeine metabolism.

Metabolism of dextromethorphan in human liver microsomes: a rapid HPCL assay to monitor cytochrome P450 2D6 activity.

A new HPLC assay was developed to study dextromethorphan O-demethylation to dextrorphan in vitro using human liver microsomes to investigate the activity of the polymorphic monooxygenase cytochrome P450 2D6 (CYP 2D6). The separation of dextromethorphan and its main metabolite dextrorphan was performed on a polymeric C18 reversed-phase column with UV-detection using levallorphan as an internal standard. Liver samples from ten subjects were screened for dextrorphan formation whereby three groups with different abilities to metabolize dextromethorphan could be found. Seven microsomal preparations from extensive metabolizers showed an average dextrorphan formation rate of 298 +/- 68 pmol/mg protein.min, one sample was classified to belong to an intermediate dextromethorphan metabolizer (79 pmol/mg protein.min), whereas two samples of poor metabolizers exhibited significantly lower rates of dextromethorphan metabolism with values of 11 and 27 pmol/mg protein.min, respectively. This assay permits not only a fast in vitro screening for cytochrome P450 2D6 monooxygenase activity but is also an excellent tool to determine potential drug-drug interactions with this important metabolizing enzyme.

Determination of the new MDR-modifier PFP 6 and its metabolites in human liver microsomes by high-performance liquid chromatography.

A specific and sensitive method for the determination of PFP 6, a new compound for reverting multidrug resistance, and its two main metabolites in human liver microsomes has been developed. Samples were almost quantitatively isolated by solid phase extraction and further analysed by HPLC. Separation was achieved on an endcapped reversed phase C-18 column with triethylamine as an organic modifier in the mobile phase. The method exhibits high recovery and a good sensitivity for analyses of small sample volumes. The application of the method for screening the metabolism of this new drug in human liver microsomes is presented.