Mechanism-Based Inactivation of Human Cytochrome P450s Paul F. - - PowerPoint PPT Presentation

Mechanism-Based Inactivation of Human Cytochrome P450s

Paul F. Hollenberg Department of Pharmacology

Reductase

Cytochrome P450 (Fe 2+) Cytochrome P450 (Fe 3+)

Reductase NADPH NADP+ RCH3 O2 H2O RCH2OH

P450 Substrate Hydroxylation

S

e- O2 e- 2H+ H2O P450

Fe3+ Fe3+ Fe2+

Fe3+-O Fe2+-O2 Fe2+-O2

• S

S S S S S H H H H H H OH

P450 P450 P450 P450 P450

Terminology:

• suicide inactivator
• enzyme-activated irreversible inhibitor
• time-dependent inhibitor

Definition: A substrate that in the process of catalytic turnover is metabolized to a reactive intermediate which inactivates the enzyme.

Mechanism-Based Inactivator

P450 P450 P450

+

P450

S S

P450

P + + I

P450 I P450

I + P

Mechanism-Based Inactivators

Enzyme substrates Require all coenzymes and substrates Activity loss is first-order with enzyme Exhibit saturation kinetics Inactivation is stoichiometric GSH and DDT do not protect against inactivation Inactivation is irreversible

Fe Cys I Fe Cys I Fe Cys I

Three Pathways for Mechanism-Based Inactivation Apoprotein Heme Crosslinked

Information that Can be Obtained with Mechanism-Based Inactivators:

Structural Studies

a) Site of adduct binding:

• heme
• protein
• i.d. adducted peptide
• i.d. adducted amino acid

b) site-directed mutagenesis

Mechanistic Studies

a) Identify the step(s) in the P450 reaction that are compromised and result in the loss in activity

0 min 5 min 10 min 15 min

Primary Reaction P450 Reductase NADPH Inactivator Secondary Reaction Primary rxn mix 7-EFC NADPH HFC product formation

5 min 5 min 5 min 5 min

Method

Proposed Mechanism for Diaziridine Oxidation

R 1 R 2 NH HN R 1 R 2 NH HN

• e-

R 1 R 2 N HN bond scission

• H+
• e-, -H+
• N2

R 1 R 2 carbene R 1 R 2 H Nu-E nz insertion

R 2

C F 3

N H H N R 3 R 1

Structures of Substituted Aryl Diaziridines

(1) R1 = H, R2 = OCH3, R3 = H (2) R1 = H, R2 = OCH2CH3, R3 = H (3) R1 = H, R2 = OCH3, R3 = OCH3 (4) R1 = H, R2 = OCH3, R3 = CH3 (5) R1 = OCH3, R2 = OCH3, R3 = OCH3 (6) R1 = H, R2 = SCH3, R3 = H

Inactivation of P450 2B6 by the Substituted Aryl Diaziridines

Substitution P450 2B6 4-methoxy (1) 65 % 4-ethoxy(2) 62 % 3,4-dimethoxy(3) 70 % 3-methyl,4-methoxy (4) 70% 3,4,5-trimethoxy (5) 4-methylthio (6) 70 % No loss Activity Loss (% of Control)

No inactivation was observed with P450s 2C9, 2D6, 2E1, or 3A4

Time- and Concentration Dependent Inactivation of P450 2B6 by 3- (Trifluoromethyl)-4-methoxy(3-methylphenyl)diaziridine

0.0 2.5 5.0 7.5 10.0 12.5 15.0 1.5 1.6 1.7 1.8 1.9 2.0

Time (min) log % Activity Remaining

• 0.5

0.0 0.5 1.0 1.5 2.0 25 50 75 100 125 150

1/[S](μM-1) 1/kobserved

(min)

Kinetic Parameters for Inactivation of P450 2B6 by the Substituted Aryl Diaziridines

Substituted aryl diaziridine KI µM kinact min-1 t1/2 min 4-methoxy (1) 7.1 ± 1.9 0.042 16.5 4-ethoxy (2) 2 ± 0.7 0.079 8.8 3,4-dimethoxy (3) 2.5 ± 1.2 0.06 11.4 3-methyl,4-methoxy (4) 1.7 ± 0.2 0.066 10.5 3,4,5-trimethoxy (5) 4-methylthio (6) 2.7 ± 0.9 0.05 No inactivation 14

Partition Ratios for the Inactivation of P450 2B6 by the Substituted Aryl Diaziridines

Substituted aryl diaziridine 4-methoxy (1) 4-ethoxy (2) 3,4- dimethoxy (3) 3-methyl,4- methoxy (4) 3,4,5- trimethoxy (5) Partition Ratio 41 62 9.6 29 45

Other Properties for the Inactivation of P450 2B6 by the Substituted Aryl Diaziridiens Addition of reductase to the inactivated protein does not lead to recovery of activity Inactivation is irreversible There is no significant heme modification 10 mM GSH does not protect against inactivation

Structures of the Aryl Diazidirines

NH N H CF3 O C H3 NH N H CF3 O C H3 NH N H CF3 O C H3 O CH3 NH N H CF3 O C H3 CH3 NH N H CF3 S C H3 NH N H CF3 O CH3 NH N H CF3 Cl NH N H CF3 NH N H CF3 O O O C H3 C H3 CH3 CF3 O O C H3 NH N H CF3 O C H3 D D D D

1 2 3 4 5 6 7 8

9

10 11

Metabolic Stability of the Aryl Diaziridines

20 40 60 80 100

1 6 7 8 9 10 % Remaining Aryldiaziridines

GC-MS Spectrum of the Metabolite of Aryl Diaziridine 1 (a) and its Ketone Standard (b)

40 80 120 160 200 240

m/z Abundance

43 7792 107 135 204 64

(a)

40 80 120 160 200 240

m/z

50 64 7792 107 135 204

(b)

50

Metabolism of an Aryl Diaziridine to a Ketone

N H N H CF 3 O C H 3

P450 NADPH

CF 3 O O C H 3

LC-MS/MS Analysis of GSHEE Adducts of Aryl Diaziridine 1

200 300 400 500 m/z 381 190 407 207 200 300 400 500 m/z 381 190 407 207

N H S O E tO O C N H N H

2

H O O C O O H F F F +2H 381 407 +2H 207 190

20 24 28 Time (min) 20 24 28 Time (min)

Proposed Chemical Structures for the GSHEE-Adducts formed by P450 2B6

NH N H CF3 O C H3 NH N H CF3 O C H3 NH N H CF3 O C H3 O CH3 NH N H CF3 O C H3 CH3 NH N H CF3 O O O C H3 C H3 CH3

CF3 OH CF3 OH CF3 O C H3 OH CF3 OH CH3 CF3 OH O O CH3 CH3

P450 2B6 NADPH GSHEE 1 2 3 4 5

SGEE SGEE SGEE SGEE SGEE

m/z 200 300 400 500 385 190 411 207 m/z 200 300 400 500 385 190 411 207

N H

S

O E tO O C N H NH

2

H O O C O O H F F F D D D D +2H 385 411 +2H 207 190

20 24 28 Time (min) 20 24 28 Time (min)

LC-MS/MS Analysis of GSHEE Adducts

• f Aryl Diaziridine 11

Proposed Mechanism for the Inactivation of P450 2B6 by Aryl Diaziridines 1-5

NH N H CF 3 OR R' R" N N H CF 3 OR R' R" N CF 3 OR R' R" NH OH O CF 3 OR R' R" C N CF 3 OR R' R" NH CF 3 O R' R" SG-EE CF 3 OH R' R" N CF 3 R' R" NH C OR N CF 3 R' R" N H OR OH N CF 3 O R' R" NH N CF 3 OH R' R" N H SG-EE P450 2B6 Protein [Fe-OH

3+]

P450

• N 2H 2

GSH-EE GSH-EE

• ROH

[Fe-OH 3+] P450

1 - 5 R'=R"=H: 10 17 18

inactivate

. .

• H .

P450

• N 2
• N 2

.

Pathway for the Metabolism of Compound 6 without Formation of a Reactive Intermediate

NH N H CF3 SMe N N H CF3 SMe N CF3 SMe NH OH CF3 SMe O C N SMe NH CF3 O N CF3 O N H

X

• H

P450

• N2H2

[Fe-OH 3+] P450

. . .

An Alternative Mechanism for the Inactivation of P450 2B6 by Aryl Diaziridines

O C H3 F 3C NH N H O H NH N H F 3C

• H +
• 2e-

inactivation or SG-EE conjugation

1 19 20

unstable intermediate CYP 2B6 O N NH F 3C

P450 2B6 and 4-hydroxy phenyl diaziridine

Km = 4.4µM and Vmax = 0.02

Time (min) Relative Abundance 28.19

100 150 200 250 300 350 400 450 500 550 600 m/z

335.9 509.8 Relative Abundance Relative Abundance

100 150 200 250 300 350 400 450 500 550 m/z

380.8 189.7 406.9 436.1 206.6 508.7 413.9 449.6 491.6 344.5 249.9 389.0

N H S O EtOOC NH NH 2 HOOC O OH F F F +2H 381 407 +2H 207 190

NH NH

C H3 CH3 C H3 CH

4-tert-butylphenylacetylene (BPA) MW = 158 g/mol

0.0 0.5 1.0 1.5 0.6 1.4 2.2

• 1

1 2 3 2 4 6

Time (min) Log % Activity Remaining

1/BPA (μM-1) 1/kobs (min)

Inactivator P450 KI kinact kinact / KI Partition ratio μM min-1 min-1mM-1 BPA WT 0.7 1.64 2343 1 T205A 16 0.36 23 9 BMP WT 17 0.56 33 10 T205A 16 0.14 9 35

20 40 60 80 100 20 40 60 80 100

BPA/P450 % Activity Remaining

+NADPH

50000 52000 54000 56000 58000

mass

20 60 100

55885

Control

50000 52000 54000 56000 58000

mass

20 60 100

56059

55885 Relative Abundance Relative Abundance

∆M = 174 (BPA + one oxygen)

335 353 278 250 407

Time (min)

20 25 30

25.8

m/z 200 300 400

C H3 C H3 CH3 O S NH O O N H2 O OH NH OH O

A Extracted ion chromatogram B

MS/MS spectrum m/z 482

353 407 278

• CO

250 205 205 335

• H2O

C 482 Da – 308 Da = 174 Da

Modified peptide positions and sequence Modified residue Precursor ion charge XCorr Probability

296FFAGTETSSTTLR308

Thr302 2 3.62 1.7 x 10-6

296FFAGTETSSTTLR308

Ser303 2 3.48 1.1 x 10-4

100TIAVIEPIFK109

Thr100 2 2.90 8.0 x 10-5 SEQUEST database search results Xcorr: cross-correlation value between the observed peptide fragment mass spectrum and the one theoretically predicted. Probability: scoring algorithm in BioWorks based on the probability that the peptide is a random match to the spectral data

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 100

b2 295.1 y2 288.3 b3 366.2 y3 389.4 b4 423.4 y4 490.5 b5-H2O 506.3 y5 577.5 b6-H2O 635.4 [y11]+2 649.7

y6 664.5

[y12]+2 723.4 (MH+-2H2O)+2 778.8 b7-H2O

910.5

y7 939.7

b8-H2O 997.6

y8 1068.7 b9-H2O 1084.6 y9 1169.8 b10-H2O 1185.6 y10 1226.8 y11 1297.8 b11-H2O 1286.6 b12 1417.5

Relative Abundance

[y11-H2O]+2 640.7

m/z b ion residue y ions 148.1 b1 F y13 ----- 295.1 b2 F y12 1444.6 366.1 b3 A y11 1297.6 423.2 b4 G y10 1226.5 524.3 b5 T y9 1169.5 653.3 b6 E y8 1068.5 928.3 b7 T y7 939.4 1015.4 b8 S y6 664.4 1102.4 b9 S y5 577.3 1203.5 b10 T y4 490.3 1304.5 b11 T y3 389.3 1417.6 b12 L y2 288.2

• b13 R y1 175.1

.0 .0 5 8 .0 1 .0 1 2 .0 1 4 .0 1 6 .0 1 8 .0 2 .0 .0 .0 5 8 .0 1 .0 1 2 .0 1 4 .0 1 6 .0 1 8 .0 2 .0

Time (min) 8 10 12 14 16 18 Absorbance (254 nm) Absorbance (254 nm) 0.05 0.05 16α-OH-testosterone androstenedione testosterone

Control 2B1

16α-OH-testosterone 16β-OH-testosterone androstenedione testosterone

BPA-modified 2B1

16β-OH-testosterone

Reversible Docking of BPA in the CYP2B1 Active Site

F297 F115 V367 I104 I101 I114

Modified residue location Distance to heme iron ( Å )a Distance to BPA ( Å )a Distance to testosterone ( Å )a Thr100 B’ helix/loop 15.44 8.31 6.94 Thr302 I-helix 6.22 3.42 2.42 Ser303 I-helix 8.57 7.67 7.18

aDistance between the nearest atom of each residue and the heme iron,

BPA, and testosterone based on CYP2B1 homology modeling.

B’ helix/loop region B’helix/loop region I-helix I-helix BPA Testosterone T100 T100 T302 T302 S303 S303

Time (min) 2 4 6 8 10 Log (Remaining Activity %)

• 2
• 1

1 2

tBPA (μM) 2 4 6 8 10 Vobs (min-1) 0.0 0.1 0.2 0.3

Time- and Concentration Dependent Inactivation of P450 2B4 by tert-butylphenylacetylene

[tBPA]/[P450]

1 2 3 4 5 6 7 8 9 10

Remaining Activity%

20 40 60 80 100

Partition Ratio for Mechanism-based Inactivation of P450 2B4 by tert-butylphenylacetylene

• cyt b5

+cyt b5

Mass (Da) 52000 53000 54000 55000 56000

54122 53948

5 min 10 min 15 min

P450 2B4-tBPA Adduct Formation as Revealed by LC-MS Analysis

∆M = 174 = tBPA+O

Wavelength (nm)

350 400 450 500 550 600 650 700

Absorbance

0.0 0.1 0.2 0.3 0.4 0.5 0.6

Absorbance

• 0.01

0.00 0.01 0.02 0.03 0.04 417 422 645 568 534

UV-visible Spectra of tBPA-modified P450 2B4

― P450 2B4

• P450 2B4 + BNZ
• modified P450 2B4
• modified

P450 2B4 + BNZ

Catalytic Activity of tBPA-modified P450 2B4

Substrates Relative Turnover Rates (% of unmodified 2B4) 7-EFC 30 BNZ 21 Testosterone 9.6 Compounds Volume (Å3) tBPA 198.7 7-EFC 226.6 BNZ 289.1 Testosterone 313.9

Time (s)

10 20 30 40 50

Absorbance at 450nm

0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24

Rates of Electron Transfer from P450 Reductase to tBPA-modified Ferric P450 2B4

• P450 2B4

― P450 2B4 + BNZ

• Modified P450 2B4
• modified

P450 2B4 + BNZ

Peptide Mapping to Identify Site of Covalent Binding

Molecular Modeling Showing the Binding of tBPA in the Active Site of P450 2B4

F115 I101 F297 A298 T302 E301 V477 V367 I363

Proposed Mechanism for Mechanism-based Inactivation of P450 2B4 by tert-butylphenylacetylene

Acknowledgements

• Hsia-lien Lin
• Ute M Kent
• Yoshimasa Kobayashi
• Chitra Sridar
• John M Rimoldi
• Satish G Puppali
• Haoming Zhang
• Lucy Waskell
• Daiichi Pharmaceutical Co., Ltd.
• NIH CA 16954