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P450 Reactions: The Basic, the Exotic, & the Important ......Dispos. 32, 1201-1208 & also L. C....
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P450 Reactions: The Basic, the Exotic, & the Important Prof. F. P. Guengerich Vanderbilt University School of Medicine [email protected] http://www.toxicology.mc.vanderbilt.edu http://isihighlycited.com/author.cgi?link1=Browse&link2=Results&id=197 Gerald T. Miwa Symposium 9 April 2007
Transcript of P450 Reactions: The Basic, the Exotic, & the Important ......Dispos. 32, 1201-1208 & also L. C....
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P450 Reactions:The Basic, the Exotic, &
the Important
Prof. F. P. GuengerichVanderbilt University School of Medicine
[email protected]://www.toxicology.mc.vanderbilt.edu
http://isihighlycited.com/author.cgi?link1=Browse&link2=Results&id=197
Gerald T. Miwa Symposium9 April 2007
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Guengerich (2003) Molec. Interventions 3, 194-204
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Guengerich (2005) in Cytochrome P450, 3rd Ed.(Ortiz de Montellano, ed.), Kluwer-Plenum, NY, pp. 377-531
Classification of Human P450s Based on Major Substrate Class
Sterols Xenobiotics Fatty acids Eicosanoids Vitamins Unknown
1B1 1A1 2J2 4F2 2R1 2A7
7A1 1A2 4A11 4F3 24 2S1
7B1 2A6 4B1 4F8 26A1 2U1
8B1 2A13 4F12 5A1 26B1 2W1
11A1 2B6 8A1 26C1 3A43
11B1 2C8 27B1 4A22
11B2 2C9 4F11
17 2C18 4F22
19 2C19 4V2
21A2 2D6 4X1
27A1 2E1 4Z1
39 2F1 20
46 3A4 27C1
51 3A5
3A7
“Orphans”
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J. A. Williams et al. (Pfizer) (2004) Drug Metab. Dispos. 32, 1201-1208& also L. C. Wienkers & T. G. Heath (Amgen) (2005) Nature Rev. Drug Discov. 4, 825-833
Fractions of drugs metabolizedvia each enzyme system
Fractions of drugs metabolizedvia each P450
P450
3A4
2D6
2C9
1A2
2C19
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The Basic
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Fe3+
Fe3+ RH
Fe2+ RH
Fe2+-O2 RH
Fe2+-O2- RH
Fe-OOHRH
FeO3+ RH
FeOH3+ R•
Fe3+ ROH
H+ 1e-
1e-
RH-ROH1.
2.
3.
4.5.
6.
7.
8.
9.
O2
NADPH-P450 reductasered
NADPH-P450 reductaseox
NADPH-P450 reductasered
NADPH-P450 reductaseox
-H2O
-
CHC HOC
N CH 2 R N CH 2 R
N CHR N CHR
N CHR NH CHROH
O
X X XO
R
R
RO
OFe 4+
HO
N
R
NN
O
R
R
R
HO
N
HeteroatomOxygenation:
HeteroatomRelease:
CarbonHydroxylation:
Epoxidation and GroupMigration:
•
•• •
• ••
•• •• +
+
•
•• •+
+-
•+
•
N
N
N
N
[FeO] 3+ [FeOH] 3+ Fe 3+
[FeO] 3+ [FeO] 2+
[FeOH] 3+
Fe 3+
[FeO] 3+ [FeO] 2+ Fe 3+
[FeO] 3+
[FeO] 2+
Guengerich & Macdonald (1984) Acct. Chem. Res. 17, 9-16
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Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
Fe3+
Fe3+S
ΔG
Fe2+S
Fe2+•O2S
Fe+•O2S
FeO3+S Fe3+
P
Fe3+
P450 ReactionsO
R2
R1
R3
O
R2
R1X R3
R2
R1O
HC CH
R3
R2R1
H
OR OR
OR
O
OH
O
R2R1 R1
R2
O CH 3 O
NH
N
NH2
N
N
NH2
RO
H R
RC N
OHX OHHO
R2R1
R3
OHR2
R1
R3
OH
RO PO
OO
NMe 3RO P
O
OO- HO NMe 3
RH ROH
O
O R2
R1
RX RX—O+
OR2
R1O
+ HXR3
RX R• + X-
+•
R2
R1
+ RCHO
+
R2
O
R1
HO
+ HCO 2H
RCH=N-OH
-X-
+
OHC CHO
+ +
+
-
-
Fe3+
Fe3+ RH
Fe2+ RH
Fe2+-O2 RH
Fe2+-O2- RH
Fe-OOHRH
FeO3+ RH
FeOH3+ R•
Fe3+ ROH
H+ 1e-
1e-
RH-ROH1.
2.
3.
4.5.
6.
7.
8.
9.
O2
NADPH-P450 reductasered
NADPH-P450 reductaseox
NADPH-P450 reductasered
NADPH-P450 reductaseox
-H2O
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Step 3 of aromatization of androgens by P450 19A1:
Akhtar et al. (1982) Biochem. J. 201, 569-580Cole & Robinson (1988) J. Am. Chem. Soc. 110, 1284-1285
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Step 3 of aromatization—FeO3+ alternative:
Hackett et al. (2005) J. Am. Chem. Soc. 127, 5224-5237
FeO3+ + H• =
-
k~ 106 M-1 s-1
kon = 2.7 x 106 M-1 s-1koff = 5.7 s-1
kcat= 0.17 s-1
(kform= 0.05 s-1)
P450 2A6 coumarin 7-hydroxylation
k= 7.5 s-1 + 0.13 s-1
kon = 1.5 x 106 M-1 s-1koff = 36 s-1
Kd = 24 µM
koff = 6.8 s-1
k= 0.3 s-1
(kform= 0.05 s-1)
(kform= 0.17 s-1)
rate-
limiting
Yun et al. (2005) J. Biol. Chem. 280, 12279-12291
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5-benzyloxyindolecoumarin
Wu, Podust, & Guengerich (2005) J. Biol. Chem. 280, 41090-41100
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Yano et al. (2004) J. Biol. Chem. 279, 38091-38094Williams et al. (2004) Science 305, 683-686
350 400 450 500
ΔA = 0.01
Wavelength, nm0 50 100 150 200
0
0.03
0.06
[Testosterone], µM
350 400 450 500
ΔA = 0.02
Wavelength, nm0 20 40 60
0
0.05
0.10
[αNF], µM
C
D
A
B
. .
A
0 20 40 60 800
2
4
6
8
0 20 40 60 80 0
0.2
0.4
0.6
0.8
1.0
[AFB1], µM
v, n
mol
pro
duct
form
ed m
in-1
(nm
ol P
450)
-1
8,9-
epox
idat
ion
B
3α-h
ydro
xyla
tion
Ueng et al. (1997)Biochemistry 36, 370-381
Hosea et al. (2000)Biochemistry 39, 5929-5939
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Krauser & Guengerich (2005) J. Biol. Chem. 280, 19496-19506
OH
OHβHα
P450
3A4
OH
OOHH
6
P450 3A4 abstracts H and rebounds O only at the (6)β face
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Ekross & Sjögren (2006) Proc. Natl. Acad. Sci. USA, in press
Ketoconazole (X2) Erythromycin
Binding of P450 3A4 to two substrates
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P450 3A4 (testosterone)
Isin & Guengerich (2006) J. Biol. Chem. 281, 9127-9136
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Crystal Structure of P450 3A4 Bound to Progesterone
Williams, P.A. et al. (2004) Science 305, 683-686
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Pyrene 1-OH Pyrene
OH
Pyrene eximer complex
O
O
α-Naphthoflavone(α-NF)
O
OO
5,6-epoxide
H+
O
OHO
OH
Rabbit P450 1A2
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Binding of 4-isopropoxynitrobenzene to(Fe3+) rabbit P450 1A2
Δ Absorbance slow & biphasic
1,4-IPNB Binding Kinetics to Rabbit P450 1A2
0 20 40 600.000
0.005
0.010
0.015
0.020
0.025Syringe 2: 2 μM P450only
[1,4-IPNB]f = 10 μM
time, s0 1 2 3 4 50.000
0.005
0.010
0.015
0.020
0.025
time, s
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Stopped -flow Fluorescence(Pyrene binding to rabbit P450 1A2)
0 1 2 3 40.50
0.55
0.60
0.65400 nm bandpass>455 nm long pass
[Pyrene]f =2 μM[P450]f = 1 μM
time, s
MonomerExcimer
0.0 0.5 1.0 1.5 2.0 2.5 3.00.50
0.51
0.52
0.53
time, s
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Human P450 1A2•αNF (2007) E. F. Johnson, JBC, in press
[O]
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The Exotic
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Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
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Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Hecker & Ullrich (1989) J. Biol. Chem. 264, 141-150Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
Prostaglandin rearrangements (P450s 5A1, 8A1)
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Chang et al. (1996) Biochemistry 35, 464-471Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
More hydroperoxide rearrangements
Plant P450
Rat P450 2B1
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Itoh & Howe (2001) J. Biol. Chem. 276, 3620-3627Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
Formation of enol ether by rearrangement
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Reductive activation reactions
Stiborova et al. (2001) Chem. Res. Toxicol. 14, 1128-1137Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring ExpansionBaeyer-Villiger Chemistry (?) in Natural Product Biosynthesis
Udwary et al. (2002) J. Am. Chem. Soc. 124, 5294-5303Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring ExpansionBaeyer-Villiger Chemistry (?) in Natural Product Biosynthesis
Kim et al. (2005) Plant Cell 17, 2397-2412Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring contraction reactions
Yin et al. (2004) Biochemistry 43, 5455-5466Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Guengerich (1989) J. Biol. Chem. 264, 17198-17205
He et al. (1989) J. Biol. Chem. 280, 22697-22705
P450 4A1
Halogen oxygenation
P4502B1
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Halogens:Oxidative dehalogenation of an aryl halideP450 101A1 (P450cam)
Chen et al. (2002) J. Biol. Chem. 277, 37519-37526Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Oxidative aryl migration in P450 93C-catalyzed isoflavone biosynthesis
Hashim et al. (1990) FEBS Lett. 271, 219-222Isin & Guengerich (2006) Biochim. Biophys. Acta, in press
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Williams et al. (1994) Carcinogenesis 15, 2733-2738Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
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Gillam et al. (1999) BBRC 265, 469-472
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1 2 3 4 5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 20
21 22 23 24 25
26 27
A WT P450 2A6
B L240C/N297Q
C N297Q
D F209T
E L240C
F N297H
Patternfor wells:
Production of colored products from substituted indolesby P450 2A6 & mutants (in vivo)
Nakamura et al. (2001) Arch. Biochem. Biophys. 395, 25-31
5-OMe indole
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Chem. Eng. News (2001) Jan. 8, p. 30
See also June 2003 National Geographic(& April 2004 Discovery Magazine)
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Coupling: Reactions outside the P450 active site
5-benzyloxyindole
Wu, Podust, & Guengerich (2005) J. Biol. Chem. 280, 41090-41100
Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Plant Biotechnol J. 2007 Jan;5(1):185-189
Warzecha H, Frank A, Peer M, Gillam EM, Guengerich FP, Unger M.Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.
(BX1 = indole synthase, which liberates indole from indole 3-glycerolphosphate;indican = indole 3-O-glucose)
Culture: Plants:
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The Important
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Activation of troglitazone: 2-sided pathway
Yamazaki et al. (1999) Drug Metab. Dispos. 27, 1260-1266Kassahun et al. (2001) Chem. Res. Toxciol. 14, 62-70Reddy et al. (2005) Chem. Res. Toxicol. 18, 880-888Isin & Guengerich (2006) Biochim. Biophys. Acta 1770, 314-329
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Coupling:Reactions in the P450 active site (?)
• = Carbons to be coupled
Ammann et al. (1995) Heterocycles 40, 425-440Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
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Pylypenko et al. (2003) J. Biol. Chem. 278, 46727-46733Zerbe et al. (2004) Angew. Chem., Int. Ed. Engl. 43, 6709-6713Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Reactions in the P450 active site (?)
P450
1A1
Totsuka et al. (1998) Carcinogenesis 19, 1995-2000Guengerich (2001) Chem. Res. Toxicol. 14, 611-650
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Ring formation reactions
Lee et al. (2004) Rapid Commun. Mass Spectrom. 18, 1901-1910Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring formation reactions
Egger et al. (1988) Drug Metab. Dispos. 16, 568-575Doss et al. (2005) Chem. Res. Toxicol. 18, 271-276Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring formation reactions
Marchetti et al (1973) Arzneim. Forsch. 23, 1291-1295Maurer & Kleff (1988) Arzneim. Forsch. 38, 1843-1845Dalvie et al. (2002) Chem. Res. Toxicol. 15, 269-299Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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Ring formation reactions
Zhang et al. (2005) Chem. Res. Toxciol. 18, 675-685Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
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k~ 106 M-1 s-1
kon = 2.7 x 106 M-1 s-1koff = 5.7 s-1
kcat= 0.17 s-1
(kform= 0.05 s-1)
P450 2A6 coumarin 7-hydroxylation
k= 7.5 s-1 + 0.13 s-1
kon = 1.5 x 106 M-1 s-1koff = 36 s-1
Kd = 24 µM
koff = 6.8 s-1
k= 0.3 s-1
(kform= 0.05 s-1)
(kform= 0.17 s-1)
rate-
limiting
Yun et al. (2005) J. Biol. Chem. 280, 12279-12291
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SuperoxideO2 + e O2-
reactive but highly selective Hydrogen peroxide
O2- + 2e + 2H+ H2O2relatively unreactive
Hydroxyl radicalH2O2 + e OH- + ●OHhighly reactiveOnly local damage
Valko et al. (2006) Int. J. Biochem. Cell Biol. 39, 44-84
P450?
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Valko et al. (2006) Int. J. Biochem. Cell Biol. 39, 44-84
4
4
4 4
4
-
Microsomal P450 as a potential source of ROS:1A (Slezak et al. 1999; Nebert et al. 2000; Delescluse et al. 2001; Twaroski et al. 2001; Liu
et al. 2001; Senft et al. 2002; Dalton et al. 2002; Shertzer et al. 2004) 2B (Liu et al. 2002; Tong et al. 2003; Imaoka et al. 2004)2E (Persson et al. 1990; DuPont et al. 2000; Caro et al. 2004; Bai et al. 2006) 3A (Montolia et al. 1995; Cederbaum et al. 2006; Robertson et al. 2001)4A (Robertson et al. 2000)
Almost all of the studies have been done in vitro
Many contradictions between studies are found
Potential link between P450 inducers and oxidative stress has not been
explored well in vivo
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Hydrogen peroxide
Methyl Cellosolve (MC)
1-ABT p=0.036
Corn oil (CO)
Aroclor 1254
p=0.017
BNF p=0.15
Clofibrate p=0.038
Water
Phenobarbital (PB)
p=0.002
Isoniazid (INH)
p=0.27
**p
-
Malondialdehyde
MC
1-ABT p=0.012
CO
Aroclor p
-
Isoprostanes F2 in liver samples
MC
ABT p=0.003
CO
Aroclor p
-
MC
ABT p=0.002
CO
Aroclor p
-
This study compares influence of P450 inducers on oxidative stress both in vitro and in vivo
The P450 inhibitor 1-ABT significantly decreased markers of oxidative stress both in vitro and in vivo
Barbiturate-type P450 inducers significantly increased markers of oxidative stress both in vitro and in vivo (2B1 or 3A enzymes?)
Study with PCN—>no change in liver isoprostanes!
Several other P450s do not produce much oxidative damge in rats in vivo, incl. PPARα agonists & 2E1 inducers
CONCLUSION Summary & future experiments
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Guengerich (2001) Chem. Res. Toxicol. 14, 611-650Isin & Guengerich (2006) Biochim. Biophys. Acta 177, 314-329
Fe3+
Fe3+S
ΔG
Fe2+S
Fe2+•O2S
Fe+•O2S
FeO3+S Fe3+
P
Fe3+
P450 ReactionsO
R2
R1
R3
O
R2
R1X R3
R2
R1O
HC CH
R3
R2R1
H
OR OR
OR
O
OH
O
R2R1 R1
R2
O CH 3 O
NH
N
NH2
N
N
NH2
RO
H R
RC N
OHX OHHO
R2R1
R3
OHR2
R1
R3
OH
RO PO
OO
NMe 3RO P
O
OO- HO NMe 3
RH ROH
O
O R2
R1
RX RX—O+
OR2
R1O
+ HXR3
RX R• + X-
+•
R2
R1
+ RCHO
+
R2
O
R1
HO
+ HCO 2H
RCH=N-OH
-X-
+
OHC CHO
+ +
+
-
-
Congratulations to Gerald Miwa!
It’s been quite a ride!
-
It’s not just a lab—it’s a fraternity!
Also thanks to($) NIH R37 CA090426 & P30 ES00267
KatarinaStark
Goutam Chowdhury
Emre Isin
MarthaMartin
Jeong-YunChoi
RaySanchez
Zhong-LiuWu
Christal Sohl
P450 Reactions:�The Basic, the Exotic, & the Important�The BasicCrystal Structure of P450 3A4 Bound to ProgesteroneThe ExoticThe Important
