Structure and Function of

Cytochromes P450 2B and 3A:

From Mechanism-Based

Inactivators to X-Ray Crystal

Structures and Back

James R. HalpertSkaggs School of Pharmacy & Pharmaceutical

Sciences

University of California, San Diego

To determine the structural basis of mammalian P450 specificity in order to rationalize and predict metabolism of xenobiotics

Tools (1979) Tools (2010)

Purified rat and rabbit hepatic P450s Heterologously expressed

Chemical modification mammalian P450s

Absorbance spectroscopy Site-directed mutagenesis

Functional assays X-ray crystallography

NMR

Homology models

Molecular dynamics simulations

Pressure-perturbation spectroscopy

Isothermal titration calorimetry

Hydrogen-deuterium exchange

FRET

Long-term Objective

Goals of Predictive Drug

Metabolism

• Overcome metabolic lability of a new

chemical entity (NCE)

• Avoid drug interactions caused by an

NCE

• Predict functional consequences of

SNP's (individuals, populations)

• Predict pharmacokinetics of an NCE

Overview

1. Species, strain, and individual

differences in P450 2B function

2. Conformational plasticity of cytochromes

P450 2B

3. Multiplicity of cytochromes P450 3A in

various species

4. Cooperativity of human P450 3A4

5. Lessons learned

Species, Strain, and Individual

Differences in P450 2B Function

• Mechanism-based inactivation

• Steroid hydroxylation

• PCB oxidation

P450 2B Enzymes

1. Highly inducible by phenobarbital in a number of species (human, rat, rabbit, mouse, dog)

2. Among the first P450s purified and characterized (LM2, PB-B/P450b)

3. Metabolize angular, medium-sized neutral or basic compounds

4. Exhibit marked species and strain differences in function

• Chloramphenicol is converted by P450 2B1 to an oxamyl chloride, which binds to the ε-amino group of one or more lysine residues in the enzyme.

• The modified enzyme is unable to undergo enzymatic reduction by NADPH-cytochrome P450 reductase.

Halpert, J. (1981). Covalent modification of lysine during the suicide inactivation

of rat liver cytochrome P-450 by chloramphenicol. Biochem. Pharmacol.

30:875-881.

TIME (min)

14

C(c

pm

)

Halpert, J., Miller, N., and Gorsky, L. (1985). On the mechanism of the

inactivation of the major phenobarbital-inducible isozyme of rat liver

cytochrome P-450 by chloramphenicol. J. Biol. Chem. 260:8397-8403.

• Chloramphenicol inactivates three (2B1, 2C6, 2C11) of eight

major rat liver microsomal cytochrome P450 enzymes.

Halpert, J., Balfour, C., Miller, N.E., Morgan, E.T., Dunbar, D., and Kaminsky,

L.S. (1985) Isozyme-selectivity of the inhibition of rat liver cytochromes P-450

by chloramphenicol in vivo. Mol. Pharmacol. 28:290-296.

• N-(2-p-nitrophenethyl)chlorofluoroacetamide is a

selective inactivator of P450 2B1 in vitro and in vivo.

Halpert, J., Jaw, J.-Y., Balfour, C., and Kaminsky, L.S. (1990). Selective

inactivation by chlorofluoroacetamides of the major phenobarbital-inducible

form(s) of rat liver cytochrome P-450. Drug Metab. Dispos. 18:168-174.

• A Gly-478 to Ala substitution in P450 2B1 from Wistar-Munich rats is responsible for the low androgen 16 β-hydroxylase activity and lack of susceptibility to inactivation by N-(2-p-nitrophenethyl)-chlorofluoroacetamide.

Kedzie, K.M., Balfour, C.A., Escobar, G.Y., Grimm, S.W., He, Y.-A., Pepperl, D.,

Regan, J., Stevens, J.C., and Halpert, J.R. (1991). Molecular basis for a

functionally unique cytochrome P450IIB1 variant. J. Biol. Chem. 266:22515-22521.

WM IIB1

1616

% 1

6-H

ydro

xyla

se A

ctivity

• Canine cytochrome P450 2B11 is responsible for the hydroxylation of 245-HCB in liver microsomes from control and phenobarbital-treated animals.

• The high constitutive expression and activity of this enzyme account for the unique ability of dogs to eliminate 245-HCB in vivo.

Duignan, D.B., Sipes, I.G., Leonard, T.B., and Halpert, J. (1987). Purification

and characterization of the dog hepatic cytochrome P-450 isozyme

responsible for the metabolism of 2,2', 4,4', 5,5'-hexachlorobiphenyl. Arch.

Biochem. Biophys. 255:290-303.

24

5-H

CB

Me

tab

olis

m

(% o

f C

on

tro

l)

• Substrate recognition sites proposed on the basis of multiple sequence alignments of P450 family 2 enzymes with P450 101 can be used to help pinpoint active site residues.

• Residues 114, 290, and 363 in P450 2B11 all contribute to the unique ability to hydroxylate 245-HCB.

Hasler, J.A., Harlow, G.R., Szklarz, G.D., John, G.H., Kedzie, K.M., Burnett,

V.L., He, Y.-A., Kaminsky, L.S., and Halpert, J.R. (1994). Site-directed

mutagenesis of putative substrate recognition sites in cytochrome P450 2B11.

Importance of amino acid residues 114, 290, and 363 for substrate specificity.

Mol. Pharmacol. 46:338-345.

WT V114I D290I L363V

Enzymes

2-OHPenCB 3-OHHexCB 2-OHHexCB

nm

ol./h

r/nm

ol P

450

• A homology model of P450 2B1 based on three bacterial P450 x-ray crystal structures can be used to explain alterations in steroid hydroxylation upon side-chain substitution at 10 putative active site positions.

Szklarz, G.D., He, Y.A., and Halpert, J.R. (1995). Site-directed

mutagenesis as a tool for molecular modeling of cytochrome P450 2B1.

Biochemistry 34:14312-14322.

• Selective inhibition of P450 2B4 vs. 2B5 can be rationalized with the help of homology models based on P450 2C5.

• Differences in the determinants of inhibition are caused by residue-residue interactions as well as residue-inhibitor contacts.

Inhibition of CYP2B4, 2B5 and 2B1 by phenylimidazole derivatives

Compounds

IC50

CYP2B4 CYP2B5 CYP2B1

µM

1-Phenylimidazole 0.90 4.2 0.59

1-(4-Chlorophenyl)imidazole 0.12 15.6 0.08

4-Phenylimidazole 0.49 8.4 0.26

4-(4-Chlorophenyl)imidazole 0.04 3.8 0.07

Spatzenegger, M., Wang, Q., He, Y.Q., Wester, M.R., Johnson, E.F. and Halpert,

J.R. (2001). Amino acid residues critical for differential inhibition of CYP2B4,

CYP2B5 and CYP2B1 by phenylimidazoles. Mol. Pharmacol. 59:475-484.

• P450 2B4 is an excellent candidate for crystallization trials.

Scott, E.E., Spatzenegger, M., and Halpert, J.R. (2001). A truncation of

2B subfamily cytochromes P450 yields increased expression levels,

increased solubility, and decreased aggregation while retaining function.

Arch. Biochem. Biophys. 395:57-68.

2B P450

% P

450

Extr

acte

d2B1dH 2B4dH 2B6dH 2B11dH

Conformational Plasticity of P450

2B Enzymes

• X-ray crystallography of rabbit P450 2B4

• Hydrogen-deuterium mass exchange

studies of 2B4

• X-ray crystallography of human P450 2B6

highly concentrated protein

solution

(+ salt, detergent, DTT,

EDTA, buffer . . .)

+

precipitant

(EtOH, Na citrate pH 5.5) sitting drop

vapor diffusion

Control of Stability/Solubility

Crystallization

• protein engineering:

1 10 20 30 490

2B4 MALLLAVLLA FLAGLLLLLF RGHPKAHGRL PPGP . . . AR

2B4dH MA KKTSSKGKL PPGP . . . ARHHHH

Underline indicates alteration from the native sequence.• salt • detergent

protein + precipitant

precipitant

• The engineering and purification approaches elaborated for P450

2C5 enable 2B4 crystallization.

Cytochrome P450 2B4 Dimer

molecule 1

molecule 2

• In the absence of exogenous ligand, P450 2B4 crystallized

as a dimer, which allowed trapping of an open form of the

enzyme and helps explain ligand access to the active site.

Location of Active Site Residues in the 2B4 Open

Conformation vs. a Closed 2B5 Structure

2B4 2C5-DMZ

active site

residues

Goal: Substrate/Inhibitor-Bound Complex

Approaches to Discourage Homodimer Formation:

1. Protein engineering: H226Y

2. Choose soluble, high-affinity ligand:

3. Form protein-ligand complex early by adding inhibitor prior to

protein concentration

Cl

N

N

Cytochrome P450 2B4

Comparison of 2B4 Structures

B'-C helices

F'-G helices

2B4(H226Y)-4(4Cl)PI

2B4

Conclusions: Conformational Shift May Accommodate Sequential Substrate Binding and

Catalysis

cleft closed for regio- and

stereoselective metabolism

2B4(H226Y)-4(4Cl)PI

cleft open for substrate

binding/metabolite release

P450 Plasticity

Definition

• Involves the large-scale motions required forsubstrate access and product egress

• Includes the reshaping of the active site to adaptto ligands of different geometry

Questions

• Do the large conformational changes inferredfrom crystal structures occur in solution?

• What are the implications of plasticity forprediction of ligand binding?

Imidazole Inhibitors of P450 2B4

N

N

N

N1-BPI

1-PBI

N

N

1-TI

NCl

N

1-CPI

N

N

1-BI

N

N

Bifonazole

N

N

1-BHI

Cl

N

NH

4-CPI

N

N

Cl

Clotrimazole

N

N

1-(Dibiphenyl)MeI

Comparison of Five 2B4 Structures

Pivoting of the F-G Helix Casette to

Accommodate the Various

Inhibitors

• Remove aliquot for time point

• Quench on ice (0°C)

• Aliquot replicates

• Place all vials in dry ice to flash freeze

• Store samples in -80°C freezer

DX-MS Setup (Exchange)

Garcia, Pantazatos, and Villarreal. Assay and drug development

technologies (2004) 2(1): 81-91.

DX-MS Setup (MS)

Samples quickly thawed to 0°C from -80°C.

Garcia, Pantazatos, and Villarreal. Assay and drug development

technologies (2004) 2(1): 81-91.

2B4dH Exchange (1000s)B’-C Region F-G Cassette

2B4 No Ligand

B’-C Region F-G Cassette

2B4 + 4-CPI

B’-C Region F-G Cassette

2B4 + 1-PBI

B’-C Region F-G Cassette

Difference (4-

CPI vs. No L)

Difference in # of Deuterons

Oxidation by Human P450 2B6 of

Antiplatelet Drugs

CYP2B4:Ticlopidine Complex Ribbon Diagram

F

G

A

NH2

COOH

F’

G’

B’

C

I

H

E

D

K

J

Ticlopidine

T302

E301

F206

I363

A298

F297

S210I209

V477

F115

I101

I114

V367

Ticlopidine Bound in the Active Site of P450 2B4

in the Opposite Orientation Predicted from 2B6

Structure of Ticlopidine and the % Difference

in Paramagnetic Relaxation Rate Relative to

the Slowest Proton

Crystallization of t-BPA Labeled 2B4

• Proposed mechanism of formation of tBPA-2B4 adduct (Zhang, et al. (2009), Mol Pharm)

• Collaboration with Hollenberg Lab (University of Michigan)

Molecular Replacement

• Maps generated using 2B4-1-CPI as a MR search model

• E301 is pointed out of active site.

• Density appears to agree with docking of tBPA by Zhang, et al.

E301

T302

Induced-fit (1) vs. Conformational Selection (2) Models of P450 2B4

DX-MS suggests that an open conformation of ligand-free

enzyme predominates in solution. However, we recently

solved a structure of a closed ligand-free form of P450 2B4.

Properties of Human P450 2B6

– Major catalyst of oxidation of bupropion,

cyclophosphamide, and efavirenz

– Highly polymorphic (Q172H and K262R of

particular importance)

– Represents 1-5% of total hepatic P450

– Along with CYP3A5 one of the few human

drug metabolizing P450s yet to be crystallized

– Low thermal stability

Engineering Cytochrome P450 2B6 for Enhanced

Expression and Stability

Temperature (oC)

30 35 40 45 50 55 60 65

% A

cti

vit

y

0

20

40

60

80

100

2B1dH

2B4dH

2B6dH

2B11dHx column 5 vs y column 5

x column 6 vs y column 6

x column 7 vs y column 7

x column 8 vs y column 8

Temperature (OC)

30 35 40 45 50 55 60 65 70 75

% P

450

0

20

40

60

80

100

Temperature (oC)

30 35 40 45 50 55 60 65

% A

cti

vit

y

0

20

40

60

80

100

2B1dH

2B4dH

2B6dH

2B11dHx column 5 vs y column 5

x column 6 vs y column 6

x column 7 vs y column 7

x column 8 vs y column 8

Temperature (oC)

30 35 40 45 50 55 60 65

% A

cti

vit

y

0

20

40

60

80

100

2B1dH

2B4dH

2B6dH

2B11dHx column 5 vs y column 5

x column 6 vs y column 6

x column 7 vs y column 7

x column 8 vs y column 8

P450P450 (HS, LS, P420) Activity

Tm ( C) T50 ( C)

2B1dH 61.3 0.2 52.7 0.3

2B4dH 58.9 0.3 50.8 0.4

2B6dH 48.2 0.2 45.2 0.4

2B11dH 51.4 0.3 48.4 0.2

P450 nmol/L

Rat 2B1dH 800 - 1200

Rabbit 2B4dH 300 - 500

Human 2B6dH 50 - 75

Dog 2B11dH 400 -600

Approaches to Enable

Crystallization of Human P450 2B6

• N-terminal modification and use of

chaperones

• Internal mutations to improve stability

(Y226H) and solubility (K262R)

• Different CM-resin

• Replacement of glycerol by sucrose after

chromatography

• Inclusion of facial amphiphiles during trials

P450 2B6:4-CPI Complex Structure (2 Å)

Structural Alignment of P450 2B4

and 2B6 4-CPI Complexes

G

F

CH

I

G’

AB’

F’

F

G

I

AH

E

D

C

F’

G’

2B6 = Green; 2B4 = Magenta

The RMSD in a Cα overlay of 2B6 and 2B4 is only 0.65 Å.

2B6 Active Site Density/2B6 and

2B4 Active Site Overlay

F115

I101

V104I209

F206

E301

T302

A298

F297

I114

V367

V477

L363/I363

2Fo-Fc map contoured at 1-σ

Only residue 363 and orientation of E301 differ between the active sites.

Summary

1. P450 2B4 exhibits remarkableconformational flexibility with imidazolesof different size and shape as evidencedby X-ray crystallography and solutionmethods.

2. Despite ~100 amino acid differences, theX-ray crystal structures of the 4-CPIcomplexes of P450 2B4 and 2B6 arealmost identical.

3. How do we reconcile these twoseemingly contradictory findings?

Acknowledgments

Dr. Chris Chin Dr. Eric Johnson

Dr. Sean Gay Dr. Dave Stout

You-ai He Dr. Mike Wester

You Qun He Dr. Qinghai Zhang

Dr. Santosh Kumar Dr. Sheng Li

Dr. Hong Liu Dr. Tong Liu

Dr. Keiko Maekawa Dr. Virgil Woods

Dr. B.K. Muralidhara Dr. Paul Hollenberg

Dr. Art Roberts Dr. Haoming Zhang

Dr. Emily Scott ES003619 (JRH)

Dr. Manish Shah ES006676 (JRH)

Dr. Margit Spatzenegger GM31001 (EFJ)

Ling Sun GM59229 (CDS)

Dr. Jyothi Talakad CA016954 (PFH)

Dr. Qinmi Wang

Dr. Mark White

Dr. Ross Wilderman

Dr. Yonghong Zhao

Multiplicity of P450 3A enzymes

• Purification of multiple rat P450 3A

enzymes

• cDNA cloning of two canine P450 3A

enzymes

Graves, P.E., Kaminsky, L.S., and Halpert, J. (1987). Evidence for

functional and structural multiplicity of pregnenolone-16 -carbonitrile-

inducible cytochrome P450 isozymes in rat liver microsomes.

Biochemistry 26:3887-3894.

Treatment 9,10-

Dehydro

warfarin

R-10-OH

warfarin

Adione

6 -OH

TAO

complex

None 0.04 0.22 0.20 0.11

PCN 0.21 1.26 1.03 0.77

PCN +

CAP

0.11 2.64 0.46 0.13

• Chloramphenicol exerts differential effects on four enzyme

activities considered diagnostic of the single known PCN-

inducible P450 in female rat liver microsomes.

Halpert, J.R. (1988). Multiplicity of steroid-inducible cytochromes P450

in rat liver microsomes. Arch. Biochem. Biophys. 263:59-68.

• Rat liver microsomes contain three steroid-inducible P450 forms that

cross-react immunologically. One can be distinguished from the other

two on SDS-PAGE gels, by Ouchterolony double immunodiffusion, and

by amino terminal sequence analysis.

Fraser, D.R., He, Y.Q., Harlow, G.R., and Halpert, J.R. (1999). Use of

chimeric enzymes and site-directed mutagenesis for identification of

three key residues responsible for differences in steroid hydroxylation

between canine cytochromes P-450 3A12 and 3A26. Mol. Pharmacol.

55:241-247.

• Heterologously expressed P450 3A12 and 3A26 can account for

the two proteins in dog liver microsomes recognized by antibodies

to a purifed PB-inducible canine liver P450 (PBD-1).

Complications Presented by CYP3A4• Sigmoidal steady-state kinetics.

aflatoxin B1, amitriptyline,

carbamazepine, diazepam,

17 -estradiol, progesterone,

testosterone.

• Stimulation by some substrates of the oxidation of other substrates.

-naphthoflavone, diazepam, progesterone, quinidine, testosterone.

• Partial or no inhibition in the presence of two substrates.

erythromycin and testosterone, midazolam and terfenadine, aflatoxin B1 and -naphthoflavone, progesterone and 7-benzyloxy-4-trifluoromethyl-coumarin.

Domanski, T.L,, He, Y.-A., Khan, K.K., Roussel, F., Wang, Q., and

Halpert, J.R. (2001). Phenylalanine and tryptophan scanning

mutagenesis of CYP3A4 substrate recognition site residues and effect

on substrate oxidation and cooperativity. Biochemistry 40:10150-

10106.

•The differential effects of site-specific substitutions on

multiple P450 3A4 activities and heterotropic activation by

ANF suggest the presences of three subpockets within a

single large active site.

Multiple Conformers as an Alternative toMultiple Binding Sites within One P450Molecule

Initial concept: P450 is

represented by two

conformers with different

substrate specificity and

different response upon

substrate binding (Koley

et al., 1995, 1997).

Challenge: Static distribution of the enzyme

between two populations suggested by the model

has no obvious basis.

Our Understanding of the Mechanismsof P450 3A4 Cooperativity

P450 3A4 has two binding sites for substrates revealinghomotropic cooperativity (e.g. 1-PB).

Interaction with the high affinity binding site triggers aconformational transition, which promotes binding atthe second, low affinity site and subsequent spinshift.

This second binding event is likely also accompanied by aconformational change.

P450 3A4 in solution and in the membrane is representedby a mixture of (at least) two persistent conformerswith different affinities for substrates and differentposition of spin equilibrium.

This conformational heterogeneity is stabilized by theoligomerization of the enzyme.

ANF can modulate the partitioning of these conformers.

Protein-Protein Interactions and Modulation of Drug

Metabolism, Wednesday 9:00 AM,

Anaheim Convention Center, Room 210AB

• Introduction

T. Tracy, University of Minnesota

• Effect of P450-P450 Complex Formation on Monooxygenase Function

W. L. Backes, Louisiana State University School of Medicine

• Microsomal Monooxygenase as a Multienzyme System: Exploring Protein-Protein Interactions of Cytrochromes P450

D. Davydov, University of California, San Diego

• UGT-CYP Protein Interactions: Role of Conjugating Enzymes in Modulating Oxidative Enzyme Activity

Y. Ishii, Kyushu University

• Lack of Substrate Inhibition in a Monomeric Form of Human Cytosolic Sult2A1 (Abstract 967.5)

I.T. Cook, University of Alabama at Birmingham

Acknowledgments

Dr. Dmitri Davydov

Nadia Davydova

Dr. Harshica Fernando

Dr. Jessica Rumfeldt

Dr. Elena Sineva

Dr. Tamara Tsalkova

Dr. Steve Sligar

GM054995 (JRH)

GM 31756, GM33775 (SGS)

Lessons Learned from Colleagues

• Do what you should do not just what you

can do now (EFJ, RMP).

• It’s better to be scared than bored (DCL).

• Always look out for the next generation

(MJC, RWE, BSM, PFH, AYHL, MRW,

POM)

Some Members of the P450 Superfamily