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Inhibition of Cytochromes P450 by Cyclopropylamines Molly E. Christian1,2, Shanmugam Pachaiyappan1, Emily E. Scott1, and Robert P. Hanzlik1, Department of Medicinal Chemistry, 1University of Kansas, Lawrence, KS 66045 and 2Carleton College, Northfield, MN 55057. Abstract Materials and Methods Cytochrome P450 enzymes play an essential role in drug metabolism. Oxygen atoms are inserted into P450 substrates en route to making them more hydrophilic and thus more easily excreted. Cyclopropylamines irreversibly inhibit cytochromes P450, acting as P450 substrates, but rendering the enzyme inactive through their metabolic intermediates. In order to learn more about the process through which this occurs, benzylcyclopropylamine (BCA) and cumylcyclopropylamine (CCA) were used in assays containing rat liver microsomes, which contain various types of P450 enzymes. The assays used 7-ethoxy-4-trifluoromethylcoumarin and aminopyrine as P450 substrates and measured metabolite production and thus P450 activity. • Source of P450 – rat liver microsomes – Contain several P450 isoforms, differing in amino acid sequence and substrate/inhibitor selectivity Test compound – CCA (more potent than BCA) Procedure 1) Incubate microsomes and CCA for 10 minutes 2) Pass mixture through a G-10 column to separate P450 from inhibitor 3) Measure enzymatic activity, total P450, and total protein • • Cyclopropylamine inhibitors proved to inhibit P450 activity in a time and substratedependent manner in the incubations containing microsomes, but in order to learn which specific P450s are inhibited by cyclopropylamines, these experiments and others need to be performed using reconstituted systems with individual P450 enzymes found in rat liver microsomes, such as 2C11 and 2B1. To produce P450 2C11 protein, genetic engineering was used to modify the 2C11 gene in order to increase solubility and to add a tag that will assist in purification of the protein. Insertion of the modified 2C11 gene into a plasmid will allow the gene to be expressed recombinantly in E. coli. The resulting protein can be easily purified and used in future experiments to determine which cytochrome P450 enzymes are inhibited by cyclopropylamines and the mechanism behind inhibition. This in turn will have repercussions in relation to metabolism of drugs containing cyclopropylamine substituents and intentional inhibition of P450 enzymes for reasons of drug metabolism. NcoI PCR to produce modified 2C11dH DNA fragment unmodified 2C11 gene Deletion to increase protein expression NcoI Digestion with restriction enzymes NcoI and EcoRV EcoRV EL2 plasmid 2A6dH NcoI pKK2A6dH 6038bp EFC O-deethylation P450 Substrate Cytochrome P450 enzymes are hemoproteins – Metabolize most drugs and xenobiotics according to the general formula: 7-Ethoxy-4-trifluoromethylcoumarin CF (EFC) EcoRV Digestion with NcoI NcoI and EcoRV and gel electrophoresis to isolate 4500 bp pKK___dH fragment 4607bp CH3 N P450 N OH non-enzyme R2 NH Measured metabolite + Ampicillin resistance gene Expression and purification of P450 2C11 N Scale up E. coli by growing in liquid culture R1 R1 – – H 7-Hydroxy-4-trifluoromethylcoumarin Formaldehyde (HFC) Site of drug-drug interactions in vivo when one drug inhibits the metabolism of another Cyclopropylamines, in contrast to other amines, irreversibly inactivate P450 enzymes benzylcyclopropylamine (BCA) Ion-exchange chromatography: Negatively charged carboxymethylcellulose resin binds positively charged protein while negatively charged proteins flow through. Positively charged proteins elute when a high salt buffer is added. O H HO O H O Method of Direct metabolite Fluorescence measurement/ P450 activity H 1) Nash Reagent 2) Colorimetry Figure 3-9, page 50 Stryer: Biochemistry, Fourth Edition © 1995 by W.H. Freeman and Company cumylcyclopropylamine (CCA) N H Reversibility of the effects of CCA on microsomal P450 and EFC O-deethylase Questions • • Which P450 isoforms are irreversibly inactivated by cyclopropylamines? How does inactivation occur? G-10 column nmol P450/ mg protein Activity, nmol HFC/ min/mg protein # CCA (uM) NADPH Preinc time, min 1 0 no 0 yes 2.91 100 8.96 100 2 0 no 10 yes 3.07 105 11.07 124 3 250 no 10 yes 2.69 92 9.29 104 4 250 yes 10 yes 0.84 29 0.72 8 % % Reversibility of the effects of CCA on microsomal P450 and AP N-demethylase CCA (uM) NADPH Preinc time, min 1 0 no 0 yes 3.45 100 22.7 100 2 0 no 10 yes 3.51 102 23.0 101 3 250 no 10 yes 3.49 101 22.7 100 4 250 yes 10 yes 1.02 30 5.0 22 # G-10 column nmol P450/mg proten Activity, nmol CH2O/ min/ mg protein % % CCA destroys: 1) 92% of EFC activity 2) 78% of AP activity 3) 70% of total P450 Conclusions: 1)Not all P450 isoforms in rat liver microsomes are susceptible to CCA 2)EFC and AP report and overlapping sets of P450 isoforms 3) Go to individual isoforms (2C11, 2B1) Isoforms metabolizing AP Isoforms metabolizing EFC http://www.science.smith.edu/departments/Biochem/images/8CPP_cytochrome-P450_3.jpg Cytochrome P450 Nickel affinity chromatography: Engineered histidine tag on 2C11dH protein binds to nickel while other proteins flow through column. 2C11dH protein elutes when free histidine is added. Large scale E. coli culture under conditions where P450 2C11dH protein is made Qiagen Ni-NTA Agarose Beads Handbook Figure: Interaction between nickel matrix and histidine tag. Lyse E. coli Purification of 2C11dH from E. coli contents by two different protein separation techniques Future Research Results and Conclusions N H Confirm desired plasmid by restriction enzyme digest and DNA sequencing Lyse cells and isolate plasmid DNA O O CF3 R1 EcoRV 6121bp Introduce pKK2C11dH plasmid into E. coli and grow on ampicillin-containing media N O 2C11dH AmpR http://images.spaceref.com/news/2006/DSCN2278.m.jpg R2 NcoI pKK2C11dH AmpR Aminopyrine N-demethylation Aminopyrine N O HHHH AmpR 3 O EcoRV Ligation www.bbc.co.uk/radio4/science/media/test-tubes.jpg R2 2C11dH 1514bp EcoRV 1431bp EcoRV 1524bp HHHH Histidine tag added to aid in purification NcoI 2C11dH Measuring P450 Activity Introduction • Producing individual P450s for CCA inhibition studies: Engineering P450 2C11 for high expression and easy purification Isoforms metabolizing both substrates • • Perform P450 activity assays in the presence of CCA and BCA with purified 2C11 and other P450 isoforms In addition, more substrates could be added to learn more about specific selectivity and inhibition of P450 Acknowledgements National Institute of Health Grant GM 21784. A special thanks to Yakov Koen, Patrick Porubsky, Brian Smith, Melanie Blevins, Natasha Michno, Michael Urban, and Linda Blake all of the University of Kansas for their help in the lab.