Transcript Inhibition of Cytochromes P450 by Cyclopropylamines

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)
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
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
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.