Inhibition of Cytochromes P450 by CyclopropylaminesMolly 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.

AbstractCytochrome 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

N

R1

CH3R2

N

R1

R2

OH NH

R1

R2

H

O

H

P450 non-enzyme+

NH N

H

benzylcyclopropylamine (BCA) cumylcyclopropylamine (CCA)

Questions

• Which P450 isoforms are irreversibly inactivated by cyclopropylamines?

• How does inactivation occur?

EFC O-deethylation Aminopyrine

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

O

CF3

O O

O

CF3

HO O

N

N

N

O

H H

O

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

Preinctime, min

G-10 column

nmol P450/mg

proten %

Activity, nmol CH2O/

min/ mg protein %

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

Results and Conclusions

#CCA (uM) NADPH

Preinc time, min

G-10 column

nmol P450/ mg

protein %

Activity,nmolHFC/

min/mgprotein %

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 EFC O-deethylase

Reversibility of the effects of CCA on microsomal P450and AP N-demethylase

CCA destroys:1) 92% of EFC activity2) 78% of AP activity3) 70% of total P450Conclusions: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 metabolizingboth 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

fragmentunmodified 2C11 gene

NcoI EcoRV2C11dH

1524bp HHHH

pKK2A6dH6038bp

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

AmpR

HH

HH

deletion

pKK___dH4607bp

NcoI EcoRV

AmpR

pKK2C11dH6121bp

NcoI EcoRV

AmpR

NcoI EcoRV2C11dH

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 matrixand histidine tag.

Introduce pKK2C11dH plasmid into E. coli and grow on ampicillin-containing media

Figure 3-9, page 50Stryer: Biochemistry, Fourth Edition© 1995 by W.H. Freeman andCompany

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 Universityof Kansas for their help in the lab.

Acknowledgements

http://www.science.smith.edu/departments/Biochem/images/8CPP_cytochrome-P450_3.jpg

Cytochrome P450