Orphan Cytochrome P450 & Other

Drug Metabolizing Systems 9:30 F. Guengerich (Vanderbilt Univ.)

The General Problem of Characterizing Proteins of Unknown

Function

9:50 Frank Gonzalez (Natl. Cancer Inst.)

Functional Transgenic Mouse Approaches to P450 Function

10:15 Hee-Won Park (Univ. Toronto)

Structural Biology Approaches to Function

10: 40 Zhongmei Tang (Vanderbilt Univ.)

In vitro LC-MS Approaches to Deorphanization of Cytochrome P450

Enzymes

11:05 Jeffrey Stevens (Pfizer)

Use of Humanized Mice in the Study of the Function of Human UGT

Enzymes

11:30 J. Steven Leeder (Children’s Mercy Hosp. & Clinics, Kansas City)

Determinants of Variability in Divalproex Sodium (VPA)

Glucuronidaiton in Children

The General Problem of Characterizing

Proteins of Unknown Function

ASPET/FASEB

Anaheim

25 April 2010

Prof. F. P. Guengerich

Vanderbilt University School of Medicine

Nashville, Tennessee

f.guengerich@vanderbilt.eduhttp://www.toxicology.mc.vanderbilt.edu/core_center/faculty/peter_guengerich.html

J. A. Williams et al. (Pfizer) (2004) Drug Metab. Dispos. 32, 1201-1208

& also L. C. Weinkers & T. G. Heath (Amgen) (2005) Nature Rev. Drug Discov. 4, 825-833

Fractions of drugs metabolized

via each enzyme system

Fractions of drugs metabolized

via each P450

P450

3A4

2D6

2C9

1A2

2C19

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”

Objectives and StrategiesSubstrates

& Products

Protein

DNA vector

cDNA

E.coli DH5 (F’IQ);

pCW vector; 5xHis tag;

N-terminal modification

Screen using mass spec; 18O2 etc.

(also quick trials, carcinogen activation)

Other groups; cDNA libraries;

mRNA, Gene synthesis …

Guengerich (2005) R37 CA90426-06

Wu, Bartleson, Ham, & Guengerich (2006) Arch. Biochem. Biophys. 445, 138

Wu, Sohl, Shimada, & Guengerich (2006) Mol. Pharmacol. 69, 2007

Strategies to deorphanize enzymes

• Try reactions (targeted approach)– Suggestions based on similar proteins

– Carcinogens

• Use binding strategies– High throughput spectral

– Immobilize & use crude extracts

• LC-MS searches (untargeted) with cell extracts– Differential (± enzyme, etc.)

– Isotope tags

• LC-MS searches (semi-targeted: use small libraries) ()

Mitochondrial P450s involved in Vit D3 hydroxylation

P450 27C1?

*

*

Mix: P450 27C1 or 27A1

Adrenodoxin reductase

Adrenodoxin

KPhos (7.4)

Vit D3, 25-OH Vit D3, 1-OH Vit D3,

cholesterol

NADPH-generating system

37 C, EtOAc extraction;

Evaporation;

Dissolve in MeOH

No Product !

30 min

HPLC

Wu, Bartleson, Ham, & Guengerich (2006) Arch. Biochem. Biophys.445, 138

P450s 2W1 & 2S1: umu assay

with carcingoens

Wu, Sohl, Shimada, & Guengerich (2006) Mol. Pharmacol. 69, 2007-2014

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

Rela

tive A

bu

nd

an

ce m

/z364

8.91

9.23

9.24

8.75

9.24

8.75

0

50

100

0

50

100 A

B

C

7.0 7.5 8.0 8.5 9.0 9.5 10.0

tR , min

0

20

40

60

80

100

8.91

9.23

D+ NADPH

- NADPH

50

100

4X1 bc Control

4X1 bc + NADPH

4X1 bc - NADPH

P450 4X1 and Anandamide

(Plot B & C

Together)

:

200 250 3000

4

6

8

10

Rela

tive A

bu

nd

an

ce

285.3

267.3

248.3

210.1

187.3

2

m/zStark et al. (2008) FEBS J. 275, 3706-3717

High selectivity and sensitivity

A potential approach for substrate screening

Developing untargeted approaches

Finding the proverbial needle in the a

haystack

Strategy for identifying P450 products & substrates

18OR/16OR

Finding substrates & products in crude mixtures:

subtract

With R. Sanchez, 2006

NADPH

generating

system

Purified P450

Phospholipid

NADPH-P450 reductase

Buffer

Liver extract

Vacuum/purge with Ar

10 cycles16O∕18O

1:1

Add NADPH-generating system

Incubate for 20 min at 37 oC

m∕z

M∕M+2

1:1

CH2Cl2

Extract with CH2Cl2(Derivatize, e.g. succinic anhydride)

Dry under nitrogen

LC-MS assay

Work Scheme

Sanchez-Ponce & Guengerich (2007) Anal. Chem. 79, 3355-3362

P450 3A4 + Testosterone

4 OH products

Sanchez-Ponce & Guengerich (2007) Anal. Chem. 79, 3355-3362

The most studied Streptomyces for genetic analysis and morphological changes.

– Genomic sequence data of 8 Mb S. coelicolor A3(2) were determined (2001); Sanger Centre

Streptomyces coelicolor A3(2)

Streptomyces P450 systems

• Streptomyces P450s may play important roles in biosynthetic pathways of

secondary metabolites such as antibiotics.

• Specific functions of individual P450s have been described in at least 21

different species of Streptomyces.

• Eighteen cytochrome P450 sequences were revealed from S. coelicolor

genome data by sequence annotation.

• The catalytic properties of these P450s are still not well known.

Streptomyces P450s

Strepomyces sp. P450 Secondary metabolite

produced

Function

S. griseolus CYP105A1, 105B1, 105C1

S. carbophilus CYP105A3

S. griseus CYP105D1, 105D2, 107F1

S. scerotialus CYP105D3

S. lividans CYP105D4

S.lavendulae CYP105F1, 107N1, 160A1 Complestatin Anti-HIV

S. noursei CYP105H1, 161A1 Nystatin Anti-fungal

S. tendae CYP105K1, 162A1 Nikkomycin Insecticidal

S. fradiae CYP105L1, 113B1, 154B1 Tylosin Promotant

S. clavuligerus CYP105M1

S. thermotolerans CYP107C1

S. erythraea CYP107A1, 107B1 Erythromycin Antibacterial

S. antibioticus CYP107D1 Oleandomycin Antibacterial

S. hygroscopius CYP107G1, 122A2, 122A3 Rapamycin Immunosuppressant

S. venezuelae PikC (PicK) Pikromycin Antibacterial

S. maritimus CYP107R1

S. peucetius CYP129A2, 131A1, 131A2 Daunorubicin Antitumor

S. spheroides CYP163A1 Novobiocin Antibacterial

S. avermitilis CYP171A1 Avermectin Antiparasitic

S. acidscabies TxtC Thaxtomin Phytotoxin

S. nodosus Orf1, Orf2 Amphotericin Antifungal

Biochem. Biophys. Res. Commun. 307, 610-619 (2003)

Guengerich (2002) Nature Rev. Drug Disc. 1, 359-366

Electron transport systems with

S. coelicolor P450 105D5

NADH

NADPH

Ferredoxin

Reductase

(FAD)

FDR1

FDR2

FDR3

FDR4

Ferredoxin

(FeS)

Fdx1

Fdx2

Fdx3

Fdx4

Fdx5

Fdx6

P450

105D5

fatty

acid

hydroxy

fatty

acid

Chun et al. (2007) J. Biol. Chem. 282, 17486-17500

P450 154A1

Cheng et al. (2010)

Also Prof. M. Waterman

& some ex-Dogs: R. Sanchez-Ponce, Y-J. Chun, T. Shimada

($) NIH R37 CA090426 & P30 ES00267

It’s not just a lab—it’s a fraternity!

Zhenhua Tian Qian Cheng

ZhongmeiTang

Giovanna

Salamanca-

Pinzon

Martha

Martin

Yi Xiao