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?

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