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Membrane Position of Cytochrome P450 2C9
Implications for Drug Metabolism
Karel Berka
Research Centre of Advanced Technologies and Materials, Department of
Carbon Nanostructures, Biomolecules and Hybrid Materials
and
Department of Physical Chemistry, Faculty of Science, Palacký University,
Olomouc, Czech Republic
Limoges 2014
Palacký University Olomouc
1573
RESEARCH CENTRE
OF ADVANCED TECHNOLOGIES
AND MATERIALS
www.rcptm.com
2
Metabolism of Xenobiotics
Toxin Uptake
Toxin
Phase I
enzyme Toxin-OH
Liver hepatocyte
Phase II
enzyme Toxin-
conjugate
Phase III
enzyme Excretion
[O]
Detoxification and elimination of xenobiotics is mainly done in liver
Phase I (mainly monooxygenases) convert hydrophobic chemicals into hydrophilic chemicals
Phase II (UGTs, SULTs, GSTs, NATs) further convert these products into amphiphilic anionic
conjugates
Phase III (transporters) export products out of the liver
A xenobiotic is a chemical compound which is found in an organism but which is not normally produced or expected to be present in it. - natural compounds - pollutants - drugs
Cytochromes P450
3
Cytochromes P450 (CYP)
Drug metabolism
Broad substrate specificity
=> Drug-drug interactions
Human genome - 17 CYP families, 57 isoenzymes
classification: CYP 3 A 4
family
>40% sequence
homology sub-family
>55% sequence homology
isoenzyme
*15 A-B
allele
Evans, Relling Science 286, 487,1999
Omura and Sato JBC, 239:2370,1964
4
(Anzenbacher, Anzenbacherova CMLS, 58: 737, 2001)
Acetaminophen,paracetamol
Dextromethorphan Loratidine Ropivacain Amitriptyline
Alfentanil Diazepam Losartan Sameterol Antipyrine
Alpidem Digitoxin Lovastatin Sequenavir Diclofenac
Alprazolam Diltiazem Meloxicam Sertindole Dronabinol (THC)
Ambroxol Docetaxel Methadone Simvastatin Carbamazepine
Amitriptyline 17b-estradiol Mibefradil Sulfamethoxazole Flurbiprofen
Astemizole Erythromycin Mifepristone Sulfentanil Glimepiride
Atorvastatin Ethinylestradiol N-hydroxyarginine Tacrolimus Glipizide
Benzphetamine Ethylmorphine Nevaripine Tamoxifen Glibenclamide
Bupivacaine Etoposide Nicardipine Teniposide Ibuprofen
Brotizolam Felodipine Nifedipine Terfenadine Indomethacin
Budesonide Fentanyl Niludipine Terguride Losartan
Buprenorphine Finasteride Nimodipine Terbinafine Phenytoin
Carbamazepine Flutamide Nisoldipine Testosterone Piroxicam
Citalopram Gallopamil Nitrendipine Tetrahydrocannabinol Tolbutamide
Cisapride Gestodene Omeprazole Theophylline Torsemide
Clarithromycin Granisetrone Oxodipine Tolterodine
Clozapine Haloperidol Paclitaxel (Taxol) Triazolam
Codeine Hypericum extract Pantoprazole Trimethadone
Colchicine Ifosphamide Progesterone Troglitazone
Cortisol Imipramine Propafenone Troleandomycin
Cyclobenzaprine Indinavir Proquanil Verapamil
Cyclophosphamide Irinotecan Quinidine Vinblastine
Cyclosporin A , G Ivermectin Rapamycin Warfarin (R-)
Dapsone Lansoprazole Retinoic acid (Tretinoin) Zatosetron
Dehydroepiandrosterone Lidocaine Rifabutin Zonisamide
Delaviridine Lisuride Ritonavir Zopiclone
Drugs - CYP3A4/2C9
5
Diverse superfamily of
hemoproteins
Most common reaction is a
monooxygenase reaction RH + O2 + 2H
+ + 2e– → ROH + H2O
substrates become more polar
(hydrophilic)
Buried active site Access by channels
Animal CYPs Highly promiscous (metabolize multiple
substrates)
Highly regio and stereospecific
Membrane attached (ER or mit)
Cytochrome P450 (CYP)
CYP2C9 (1OG2) Williams et al., Nature 424, 464, 2003
6
Membrane associated
(inner membranes of mitochondia, endoplasmic reticulum)
How membrane affects CYP structure?
How the membrane affects substrate binding and product release?
Role of Membrane in Substrate Binding
Black, S.D. FASEB J. 1992, 6, 680-685.
7
Literature review on
CYP Membrane Anchoring
Available structural information come from
analysis of engineered enzymes with
deleted N-terminal anchor
Several different structural models in literature
Wade, R. et al.
BBA,1754,239,2005
Orientations of Proteins in
Membranes (OPM)
Lomize, M.A.
Bioinformatics, 22:5, 2006
Zhao, Y. et al. JBC,
281:9, 2006 Williams, P. et al. Mol Cell.
2000, 5, 121 Poulos and Johnson, in. Cytochrome P450, III. Ed., 2005
Aims of the Modelling
Prepare structural model of membrane anchored CYP
Estimate penetration depth of a prototypic CYP substrate in the
membrane
Cell membrane
Environmental barrier
Proteins (up to 50%)
Lipid bilayer
Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell, 4th edition.; 2002
Singer, S. J.; Nicolson, G. L., The fluid mosaic model of the structure of cell membranes. Science 1972, 175, 720-31. 9
Four region model
Glycerophopholipis
• Phosphatidylcholine
• Phosphatidylethanolamine
• Phosphatidylserine
• Phosphatidic acid
Glycolipids
The Cell - A Molecular Approach, Cooper, Geoffrey M., Sunderland (MA) Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell, 4th edition.; 2002
Saturated fatty acids • Lauric, Myristic, Palmitic,
Stearic acid
Unsaturated fatty acids - cis • Oleic acid
Cholesterol, Sphingomyelin
Head groups region
Polar
Charge
Volume
Acyl tails regions
Nonpolar
Lenght
Saturation
[S.-J. Marrink, H.J.C. Berendsen, Simulation of water transport through a lipid membrane, J. Phys.Chem. 98 (1994) 4155–4168.]
10
11
Composition of ER Membrane
The Cell - A Molecular Approach, Cooper, Geoffrey M.,
Sunderland (MA), Sinauer Associates, Inc.; c2000
Endoplasmatic
Reticulum
Plasma
Phosphatidylcholine PhosphatidylserinePhosphatidylethanolamine SphingomyelinGlycolipids CholesterolOthers
Meer G., Voelker DR., Feigenson GW. Nat Rev Mol Cell
Biol. 2008; 9(2): 112–124.
http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An external file that holds a picture, illustration, etc.Object name is nihms89989f2.jpg [Object name is nihms89989f2.jpg]&p=PMC3&id=2642958_nihms89989f2.jpghttp://www.sinauer.com/
12
Membrane Simulations
DOPC (Dioleoylphosphatidylcholine) Berger forcefield;
Vacha et al. JPC A 2009
CYP2C9 – homology model from 1OG2
Molecular dynamics
250-ns-long MD run (2x100 + 50), NPT, 300 K, 1 atm., in explicit solvent (SPC), 90.000 UA
DOPC
13
Molecular Dynamics Simulations
Gromacs 4.0, FF G56a6 (UA)
MD run (50ns+2x100), NPT, 300 K, 1 atm., in explicit solvent (SPC), 90.000 UA
i
i
i
i
i
iiii
rr
E
tmm
rF
raF
2
2
integration
t = 2 fs
E
empirical force fields
Atomic resolution
Sub-ps time resolution Quality of force fields Huge amount of data
Data mining
14
Model Preparation
Alignment
CYP2C9
CYP2B1
CYP2B4
regular α-helix
N-terminal
transmembrane
domain (1-33) back mut. – wt-CYP2C9
System relaxes; topology of catalytic domain is saved …
Simulation results
15 Berka K, Hendrychova T, Anzenbacher P, Otyepka* M. J. Phys. Chem. A, 115(41), 11248-11255, 2011 Berka K, Paloncyova M, Anzenbacher P, Otyepka* M: J. Phys. Chem. B, 117(39), 11556-11564, 2013
16
4 nm
accessible and inaccessible epitopes -4 nm
4 nm
Experiment:Model
Epitopes
Some part of cat. domain in the membrane:β1 sheet, B/C, F‘/G‘-loops in the membrane Res. 36, 69, 380 in the membrane:36 and 69 are in the mem., 380 in head group region
Height above the membrane 3.5±0.9 nm:3.9±0.7 nm Occupied membrane area 6.8±0.95 nm2:7.9±0.6 nm2 Heme tilting angle 40-70°:35±5°
Validation step:
Comparison with experimental data
17
Where are Drugs in the Membrane?
substrate
ibuprofen
product
3-hydroxy ibuprofen
18
S = ibuprofen
P = 3-OH ibuprofen
S = ibuprofen (deprotonated)
P = 3-OH ibuprofen (deprot.)
Umbrella sampling, WHAM
Gromacs, 1 Å spacing, 2x50 windows, 5 ns each
Population of states
gives ∆G
P1/P2 = exp (∆G/kBT)
19
S = ibuprofen
P = 3-OH ibuprofen
S = ibuprofen (deprotonated)
P = 3-OH ibuprofen (deprot.)
Umbrella sampling, WHAM
Gromacs, 1 Å spacing, 2x50 windows, 5 ns each
Population of states
gives ∆G
P1/P2 = exp (∆G/kBT)
20
S=ibuprofen
P=3-OH ibuprofen
product shifted to the polar phase
Paloncyova M et al J. Phys. Chem. B, 117(8), 2403-2410, 2013
Paloncyova M et al J. Phys. Chem. B, 118 (4), 1030–1039, 2014
CYP2C9 active site is buried
21
21
Active site
Access/Egress Paths
• MOLE – more robust, quicker, overcomes some CAVER
shortcomings
– stand-alone
– online analysis, clustering etc.
– http://mole.chemi.muni.cz
• Nomenlature by Wade et al. – Cojocaru et al. BBA, 1770:390,2007
Petrek, Kosinova, Koca, Otyepka, Structure 15(11):1357, 2007
http://mole.chemi.muni.cz/
• PDB structure (PDBID,
structural assembly, own
pdb)
• Starting point
(automatic, Catalytic
Site Atlas, XYZ,
sequence, manual
selection of residues)
• Ignore (automatically
waters, possible
HETATM, or manual
selection of residues)
• Parameters (Probe
radius, Interior Threshold
radius, Origin radius,
Surface Cover radius)
http://mole.upol.cz
Berka K et al, Nuc. Acids Res., 40(W1), W222-W227, 2012
Sehnal D et al, J. Cheminform., 5, 39, 2013
de Beer TAP et al, Nucl. Acids Res. 42(D1): D292-D296, 2013
MOLEonline 2.0 – channels (input)
http://mole.upol.cz/
Visualization in Jmol
Channels (visualization, profile,
list of channel-lining residues)
Physicochemical properties
(charge, hydrophobicity,
hydropathy, polarity and
mutability)
Cavities (visualization, volume)
Molecular surface (alpha shape)
Starting points (visualization,
optimization)
Pores (merged channels,
automatic)
Re-calculation (with new
parameters)
Export (PDB , Pymol)
2j0d
CYP
3A4
MOLEonline 2.0 – channels (output)
25
Channels Found by MOLE
Channels enable substrate passage to
the deeply buried active site and
product release from the active site
26
S 2x 2x
S = ibuprofen
P = 3-OH ibuprofen
S = ibuprofen (deprotonated)
P = 3-OH ibuprofen (deprot.)
Drug CYP interaction
27
• Atomic model of CYP2C9/membrane
– agrees with most available experimental data
– consistent with interaction of CYP with CPR and cyt(b)
– TM-helix, 1 sheet, G‘ helices are in membrane
– B‘, F‘, G helices are on membrane surface
• Channels 2x (2b, 2a, 2e, 2c) - toward the membrane
– corresponds to the free energy minimum of substrate (ibuprofen)
• Solvent channel - membrane/solvent interface
– corresponds to the free energy minimum of product (3-hydroxyibuprofen)
In sum:
2x channels can be involved in substrate access,
solvent channel can be involved in product release
Summary
28
Berka K et al. J. Phys. Chem. A, 115(41), 11248-11255, 2011
29
Acknowledgements
Olomouc, CZ
Pavel Anzenbacher
Michal Otyepka
Karel Berka
Markéta Paloncýová
Ondřej Hanák (http://mole.upol.cz)
Josef Skopalík
Tereza Hendrychová
Veronika Navrátilová
Martin Šrejber
Brno, CZ
David Sehnal, Radka Svobodova Vařeková,
Jaroslav Koča (MOLE 2.0)
Martin Petřek (MOLE)
http://mole.chemi.muni.cz
Finantial Supports
CSF, Ministry of Education ...
http://mole.upol.cz/http://mole.chemi.muni.cz/http://mole.chemi.muni.cz/http://mole.chemi.muni.cz/
Thank you for your attention
• Any questions?
autor 30