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

P450 Substrate Hydroxylation RCH 3 Cytochrome Reductase NADPH P450 (Fe 2 + ) O 2 Cytochrome H 2 O Reductase NADP + P450 (Fe 3 + ) RCH 2 OH

Fe 3+ OH S H S P450 Fe 3+ -O Fe 3+ S H H S P450 P450 e - H 2 O 2H + Fe 2+ -O 2 - Fe 2+ S H S H P450 P450 Fe 2+ -O 2 H S P450 O 2 e -

Mechanism-Based Inactivator 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.

S P + + S P450 P450 P450 I I I + P450 P450 P450 + P P450

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

Three Pathways for Mechanism-Based Inactivation I I I Fe Fe Fe Cys Cys Cys Apoprotein Heme Crosslinked

Information that Can be Obtained with Mechanism-Based Inactivators: Structural Studies Mechanistic Studies a) Site of adduct binding: a) Identify the step(s) in the P450 reaction that are - heme compromised and result in - protein the loss in activity - i.d. adducted peptide - i.d. adducted amino acid b) site-directed mutagenesis

Method 5 min 0 min 5 min 5 min 5 min 10 min 15 min 5 min Primary Reaction P450 Reductase NADPH Secondary Reaction HFC product Inactivator Primary rxn mix formation 7-EFC NADPH

Proposed Mechanism for Diaziridine Oxidation H Nu-E nz HN NH HN NH HN N -e - , -H + -e - bond scission -N 2 -H + R 1 R 2 R 1 R 1 R 2 R 2 R 1 R 2 R 1 R 2 carbene insertion

Structures of Substituted Aryl Diaziridines N H H N (1) R 1 = H, R 2 = OCH 3 , R 3 = H C F 3 (2) R 1 = H, R 2 = OCH 2 CH 3 , R 3 = H (3) R 1 = H, R 2 = OCH 3 , R 3 = OCH 3 (4) R 1 = H, R 2 = OCH 3 , R 3 = CH 3 R 1 (5) R 1 = OCH 3 , R 2 = OCH 3 , R 3 = OCH 3 R 3 (6) R 1 = H, R 2 = SCH 3 , R 3 = H R 2

Inactivation of P450 2B6 by the Substituted Aryl Diaziridines Activity Loss (% of Control) 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) 70 % 4-methylthio (6) No loss 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 2.0 log % Activity Remaining 1.9 1.8 150 125 1.7 (min) 100 1/k observed 75 50 1.6 25 0 -0.5 0.0 0.5 1.0 1.5 2.0 1/[S]( μ M -1 ) 1.5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Time (min)

Kinetic Parameters for Inactivation of P450 2B6 by the Substituted Aryl Diaziridines Substituted aryl K I µM k inact min -1 t 1/2 min diaziridine 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) 2.7 ± 0.9 0.05 14 4-methylthio (6) No inactivation

Partition Ratios for the Inactivation of P450 2B6 by the Substituted Aryl Diaziridines Substituted 4-methoxy 4-ethoxy 3,4- 3-methyl,4- 3,4,5- aryl dimethoxy methoxy trimethoxy diaziridine (1) (2) (3) (4) (5) Partition 41 62 9.6 29 45 Ratio

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 H N NH H N H N NH H N NH H N NH CF 3 CF 3 CF 3 CF 3 CF 3 H 3 C O O CH 3 O H 3 C O O CH 3 O O O CH 3 H 3 C H 3 C H 3 C H 3 C 5 4 3 2 1 H N NH O H N NH H N NH CF 3 NH H N CF 3 CF 3 CF 3 CF 3 O Cl O S CH 3 H 3 C H 3 C 7 6 9 10 8 H N NH CF 3 D D D D O H 3 C 11

Metabolic Stability of the Aryl Diaziridines 100 80 % Remaining 60 40 20 0 1 6 7 8 9 10 Aryldiaziridines

GC-MS Spectrum of the Metabolite of Aryl Diaziridine 1 (a) and its Ketone Standard (b) 135 (a) (b) 135 Abundance 204 204 7792 7792 43 107 64 107 64 50 50 0 40 80 120 160 200 240 0 40 80 120 160 200 240 m/z m/z

Metabolism of an Aryl Diaziridine to a Ketone O CF 3 H N N H CF 3 P450 NADPH O O H 3 C H 3 C

LC-MS/MS Analysis of GSHEE Adducts of Aryl Diaziridine 1 O H 407 +2H 207 O 190 S E tO O C N F H F N H F O 381 381 +2H 381 N H 2 H O O C 190 190 407 407 207 207 20 20 24 24 28 28 200 200 300 300 400 400 500 500 Time (min) Time (min) m/z m/z

Proposed Chemical Structures for the GSHEE-Adducts formed by P450 2B6 5 2 3 4 1 NH H N NH H N H N NH H N NH H N NH CF 3 CF 3 CF 3 CF 3 CF 3 H 3 C CH 3 O O O H 3 C O O CH 3 O O O CH 3 H 3 C H 3 C H 3 C H 3 C P450 2B6 NADPH GSHEE CF 3 CF 3 CF 3 CF 3 CF 3 SGEE SGEE SGEE SGEE SGEE CH 3 OH O O OH OH OH O CH 3 OH CH 3 H 3 C

LC-MS/MS Analysis of GSHEE Adducts of Aryl Diaziridine 11 O H +2H 411 D D 207 O 190 D D S F E tO O C N F H N H F O +2H 385 385 385 NH 2 H O O C 190 190 411 411 207 207 20 20 24 24 28 28 200 200 300 300 400 400 500 500 Time (min) Time (min) m/z m/z

Proposed Mechanism for the Inactivation of P450 2B6 by Aryl Diaziridines 1-5 NH 1 - 5 . OH O CF 3 N H N NH H N N CF 3 CF 3 -H . CF 3 -N 2 H 2 P450 R' R" R' R" R' R" R' R" OR OR OR OR R'=R"=H: 10 NH . 3+ ] [Fe-OH P450 N CF 3 CF 3 SG-EE CF 3 17 C 18 inactivate GSH-EE P450 2B6 Protein R' R" R' R" R' R" O OH OR - N 2 - N 2 NH H N NH H N N CF 3 N CF 3 CF 3 N CF 3 N SG-EE GSH-EE [Fe-OH 3+ ] -ROH . R' R" R' R" C R' R" R' R" P450 OH OR OR OH O

Pathway for the Metabolism of Compound 6 without Formation of a Reactive Intermediate NH . OH H N N H N NH N O CF 3 . CF 3 CF 3 CF 3 -N 2 H 2 -H P450 SMe SMe SMe SMe NH [Fe-OH 3+ ] . N P450 C X SMe H N CF 3 N CF 3 O O

An Alternative Mechanism for the Inactivation of P450 2B6 by Aryl Diaziridines O H C H 3 O O inactivation or - 2e- CYP 2B6 -H + SG-EE conjugation F 3 C F 3 C F 3 C N H N NH H N NH NH 19 20 1 unstable intermediate

P450 2B6 and 4-hydroxy phenyl diaziridine

Km = 4.4µM and Vmax = 0.02

Relative Abundance 28.19 Time (min) Relative Abundance 509.8 OH 407 +2H 335.9 207 O 100 150 200 250 300 350 400 450 500 550 600 190 m/z S EtOOC N F H F NH NH F NH O +2H 381 Relative Abundance 380.8 NH 2 HOOC 189.7 406.9 436.1 206.6 413.9 491.6 508.7 344.5 389.0 449.6 249.9 100 150 200 250 300 350 400 450 500 550 m/z

CH H 3 C H 3 C CH 3 4- tert -butylphenylacetylene (BPA) MW = 158 g/mol

2.2 Log % Activity Remaining 1.4 6 1/k obs (min) 4 2 -1 0 1 2 3 0.6 1/BPA ( μ M -1 ) 0.0 0.5 1.0 1.5 Time (min)

Inactivator P450 K I k inact k inact / K I Partition ratio μ M min -1 min -1 mM -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

100 80 % Activity Remaining 60 40 20 0 0 20 40 60 80 100 BPA/P450

Control 55885 100 Relative Abundance 60 20 50000 52000 54000 56000 58000 mass 56059 100 Relative Abundance +NADPH 60 ∆ M = 174 (BPA + one oxygen) 55885 20 50000 52000 54000 56000 58000 mass

A Extracted ion chromatogram B MS/MS spectrum 335 25.8 m/z 482 353 278 250 407 205 20 25 30 200 300 400 Time (min) m/z -H 2 O 335 C 353 H 2 N O O OH NH H 3 C O S H 3 C NH OH CH 3 O O 205 407 -CO 250 278 482 Da – 308 Da = 174 Da

SEQUEST database search results Modified peptide Modified Precursor ion XCorr Probability positions and sequence residue charge 296 FFAGTE T SSTTLR 308 Thr302 2 3.62 1.7 x 10 -6 296 FFAGTET S STTLR 308 Ser303 2 3.48 1.1 x 10 -4 100 T IAVIEPIFK 109 Thr100 2 2.90 8.0 x 10 -5 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