1. Inhibition of Cytochromes P450 by Cyclopropylamines Molly E. Christian 1,2, Shanmugam Pachaiyappan 1, Emily E. Scott 1, and Robert P. Hanzlik 1, Department of Medicinal Chemistry, 1 University of Kansas, Lawrence, KS 66045 and 2 Carleton College, Northfield, MN 55057. Abstract 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. Cyclopropylamine inhibitors proved to inhibit P450 activity in a time and substrate- dependent 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. Introduction Cytochrome P450 enzymes are hemoproteins –Metabolize most drugs and xenobiotics according to the general formula: –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)cumylcyclopropylamine (CCA) Questions Which P450 isoforms are irreversibly inactivated by cyclopropylamines? How does inactivation occur? EFC O-deethylationAminopyrine N-demethylation P450 Substrate 7-Ethoxy-4-trifluoromethylcoumarin (EFC) Aminopyrine Measured metabolite 7-Hydroxy-4-trifluoromethylcoumarin (HFC) Formaldehyde Method of metabolite measurement/ P450 activity Direct Fluorescence 1)Nash Reagent 2)Colorimetry Measuring 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 Materials and Methods # CCA (uM)NADPH Preinc time, min G-10 column nmol P450/mg proten% Activity, nmol CH 2 O/ min/ mg protein% 10no0yes3.4510022.7100 20no10yes3.5110223.0101 3250no10yes3.4910122.7100 4250yes10yes1.02305.022 Results and Conclusions # CCA (uM)NADPH Preinc time, min G-10 column nmol P450/ mg protein% Activity, nmol HFC/ min/mg protein% 10no0yes2.911008.96100 20no10yes3.0710511.07124 3250no10yes2.69929.29104 4250yes10yes0.84290.728 Reversibility of the effects of CCA on microsomal P450 and EFC O-deethylase Reversibility of the effects of CCA on microsomal P450 and AP N-demethylase 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 Isoforms metabolizing both substrates Producing individual P450s for CCA inhibition studies: Engineering P450 2C11 for high expression and easy purification EL2 plasmid NcoI EcoRV Histidine tag added to aid in purification PCR to produce modified 2C11dH DNA fragment unmodified 2C11 gene NcoIEcoRV 2C11dH 1524bp HHHH pKK2A6dH 6038bp NcoI EcoRV Ampicillin resistance gene Digestion with NcoI and EcoRV and gel electrophoresis to isolate 4500 bp fragment Digestion with restriction enzymes NcoI and EcoRV Amp R HHHH deletion pKK___dH 4607bp NcoI EcoRV Amp R pKK2C11dH 6121bp NcoI EcoRV Amp R NcoIEcoRV 2C11dH 1514bp HHHH 2C11dH Ligation Deletion to increase protein expression 2A6dH 1431bp Expression and purification of P450 2C11 Scale up E. coli by growing in liquid culture Lyse cells and isolate plasmid DNA Confirm desired plasmid by restriction enzyme digest and DNA sequencing Large scale E. coli culture under conditions where P450 2C11dH protein is made Lyse E. coli Purification of 2C11dH from E. coli contents by two different protein separation techniques 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. www.bbc.co.uk/radio4/science/media/test-tubes.jpg http://images.spaceref.com/news/2006/DSCN2278.m.jpg 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. Qiagen Ni-NTA Agarose Beads Handbook Figure: Interaction between nickel matrix and histidine tag. Introduce pKK2C11dH plasmid into E. coli and grow on ampicillin-containing media Figure 3-9, page 50 Stryer: Biochemistry, Fourth Edition © 1995 by W.H. Freeman and Company Future Research 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 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. Acknowledgements http://www.science.smith.edu/departments/Biochem/images/8CPP_cytochrome-P450_3.jpg Cytochrome P450