pharmaceutical proteomics non-clinical studies subcommittee of the advisory committee for...

Pharmaceutical Proteomics

Non-Clinical Studies Subcommittee of theAdvisory Committee for Pharmaceutical Science

US Food and Drug AdministrationMarch 9, 2000

Leigh Anderson, Ph D.CEO, Large Scale Proteomics Corp.

Why Proteins?Roles of Proteome, Transcriptome, Genome

Genome (all genes): What could happen

Transcriptome (all mRNA’s): What might be happening

Proteome (all proteins): What is

happening

Molecular Technologies and Biological Function

?

??Microarrays

(Affymetrix, Incyte)

Proteomics(LSP, OGS)

Diagnosis,Pharmacology,

Toxicology

Genomics,SNPs

Protein Structure

Change (PTM)

Protein Abundance

Change

mRNA Abundance

Change

Functional Change

Genomic Difference

DNA Microarrays Will Not Suffice to Monitor Pre-Clinical Markers

• Human Liver (LSP and Incyte)

– Correlation <0.5

– Different populations of gene products

• 60 Human Cell Lines (Tew, et al.)

– Correlation = 0.43

• Human Serum Proteins (Kawamoto, et al.)

– Correlation (without albumin) is < 0

• Yeast (Gygi, et al.)

– Correlation (subset) = 0.36

Protein and mRNA Abundances Are Poorly Correlated:

• Serum

• Urine

Important Sample Types Contain No Useful mRNA

Protein (2D) vs mRNA (RTI) Abundance in Human Liver

1000 10000 100000 1000000

0.0001

0.001

0.01

0.1

1

CPSACTB

HSP60

PDI

BIP

CRTC

F1ATPB

ACTG

HSC70

CYB5

ENPL

GR75

PYVC

HSP70

TBB1

VIME

TPM

NP450R

TBA1

HSP90

COX-II

LAMR

LAMB

Protein Abundance (CB stained 2-D gel)

MessageAbundance

(% of clones in RTI)

Not detected by RTI

1 clone

2 clones

R= 0.48

LSP/Incyte 9/27/96

Replotted from data in: Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program. Tew KD, Monks A, Barone L, Rosser D, Akerman G, Montali JA Wheatley JB, Schmidt DE Jr.,

Mol Pharmacol (1996 Jul) 50(1):149-59

Protein vs mRNA for GST in 60 Human Cell Lines

0.1

1.0

10.0

100.0

1000.0

0.1 1.0 10.0 100.0

GS

T

mR

NA

(n

mo

l mR

NA

/mg

pro

tein

)

Lung

Ovarian

CNS

Leukemia

Prostate

Renal

Melanoma

Breast

Colon

R=0.43

GSTProtein (g GST/mg protein)

The protein abundance fingerprint is affected by:

• Perturbations around a “normal” state– Disease states– Treatment effects (e.g., drugs)

• Therapeutic• Toxic

• Progressive change in the “normal” state– Differentiation (e.g., cell type differences)– Evolution of the species

Drug Targets Are Proteins

The top 10 drugs in U.S. sales in 1995 bind to enzymes, receptors and channels in approximately equal proportions

Tradename Generic name$b in U.S.

sales Site of action Enzyme Receptor Channel

1 Zantac ranitidine 2.145 histamine H2 receptor X

2 Prozac fluoxetine 1.472 serotonin re-uptake ?

3 Prilosec omeprazole 1.191 H+/K+ ATPase X

4 Procardia XL nifedipine 1.101 Ca channel X

5 Epogen erythropoietin .964 epo receptor X

6 Zoloft sertraline .894 serotonin re-uptake ?

7 Vasotec enalapril .858 angiotensin converting enzyme

X

8 Mevacor lovastatin .849 HMG CoA reductase X

9 Cardizem CD diltiazem .758 Ca channel X

10 Premarin conj. estrogens .711 estrogen receptor X

Drug Mechanisms Involve Changes in Gene RegulationImplemented at the Protein Layer

Receptor Binding

SignalTransduction

Enzyme Inhibition

Drug Effects

NuclearEvents

mRNA's

Toxicology

Feedback

Proteins

NuclearEvents

mRNA'sPrecursorAccumulation

Product Deficit

Pharmacology

Toxicology

Proteins

Feedback

Pharmacology

Large ScaleProteomics

Technology for Marker Discovery

Components of the ProGEx™ Proteomics Technology Platform

• Experiment design, sample generation, fractionation and preparation

• 2-D gels: High-throughput, high-resolution

Protein “Chips”

• Mass spectrometry: high-throughput protein identification/characterization

• Software: data acquisition, DB management and mining

LSP Advanced Fractionation Technologies

• Tissue dissection

• Cell type separations

• Subcellular fractionation

• Multidimensional fractionation methods

– Chromatography

– Microbanding centrifugal methods

Annexin V

SMP-30CK-I-19

Actin ActinActin

Cholangiocytes Hepatocytes Unfract. Liver

Rare vs Predominant Cell Types in a Tissue:Cholangiocyte and Hepatocyte Protein Differences

Data from “Cholangiocyte specific rat liver proteins identified by establishment of a two-dimensional gel protein database and their role in cytoprotection”. Tietz et al. Electrophoresis, in press.

LSP 2-D Gel Technology

High-throughput• Proprietary large scale automation• Modular-replicatable systems

Reproducible• Sample preparation• Reagent standardization• Computer-controlled systems, data logging

Ultra-sensitive quantitation• Proprietary fully automated silver stain• Colloidal Coomassie Blue• Fluorescence

High Resolution• Resolution measure > 40,000• Near-gaussian spot shapes

Each spot is a distinct protein type (defined by its position)Each spot can by quantitated to yield protein abundance

A 2-D Gel is Equivalent to Thousands ofSpecific Protein Tests Done at Once

F1ATPase

Actin

Albumin

ER-60

LSP Mass Spectrometry

High-throughput MALDI• DE-STR • Automated data flow• Automated sample prep

Automated LC/MS/MS• QTof, LCQ• Sequest/Masscot searching• Sequence generation

Automated Protein Excision/Processing• 10 spots/min• Delivers to 96/384-well plates• Coupled to Kepler database• Robotic digest processing

Identification of Protein Spots by Mass Spectrometry

Scan and analyze gel Cut spots

Same gel: 200 spots cutSilver-stained gel

Digest in96-well plates

0

10000

20000

30000

40000

Co

un

ts

1000 1500 2000 2500 3000

709

.36

33

864

.35

29

977

.45

889

83.5

267

103

0.1

111

082.

495

109

8.5

071

118.

55

128

5.7

511

354.

759

138

6.8

021

430.

732

151

0.8

561

558.

805

171

1.7

53

180

6.8

761

820.

88

195

0.0

021

987.

052

207

8.0

46

225

9.1

142

321.

22

269

4.2

31

Tp

frag

men

t

MALDI-TOF LC-MS/MS + Sequest

LSP Bioinformatics

Kepler™ System• Automated image analysis• Statistical analysis• 15 years proprietary development and use

Newton™ DB Mining• Database-wide queries• Comparison of complex effects• Distance/similarity of effects

Integrated Laboratory Database

Relational Database Architecture• ~20,000 analyses currently on-line

Organization of Proteomics Results Related to Pharma Development: MAP™ and MED™ Databases

ProGEX™ Proteomics PlatformSample Generation, 2-D Gel Analysis,

Kepler Bioinformatics, MS Identification

Drug EffectsDisease ModelsKnockouts

DiseaseGenetics

Human Ex Vivo

Rodent Ex VivoHuman In Vitro

MED™Molecular Effects of

Drugs DatabaseMAP™Molecular Anatomy

and Pathology Database

DB Mining: surrogate markers, mechanisms, targets

Class Generic name Tradename

1 5-alpha-reductase inhibitor Finasteride Proscar

2 ACE inhibitor Captopril Capoten

3 Enalapril Maleate; MK421 Vasotec

4 Acne product Isotretinoin Accutane

5 Adrenal steroid inhibitors Aminoglutethimide Cytadren

6 Alzheimer's treatment Tacrine HCl Cognex

7 Analgesic Acetaminophen Tylenol

8 Androgen Stanozolol Winstrol

9 Anesthetic Halothane Fluothane

10 Anti tuberculosis Isoniazid Nydrazid

11 Antibiotic Tetracycline Hydrochloride Sumycin

12 Chloramphenicol Chloromycetin

13 Erythromycin estolate Ilosone

14 Anticonvulsant Valproic Acid Depakene

15 Antiestrogen, nonsteroidal Tamoxifen Nolvadex

16 Antifungal Ketoconazole Nizoral

17 Antineoplastic Amethopterin (MTX) Methotrexate

18 Antiviral Zidovudine (AZT) Retrovir

19 Acyclovir Zovirax

20 Ca channel blocker Amlodipine Besylate Norvasc

21 Isradipine DynaCirc

22 Verapamil HCl Calan SR

23 Carbonic anhydrase inhibitor Methazolamide Neptazane

24 Diuretic (K-sparing) Spironolactone Aldactone

25 Estrogens Conjugated estrogens Premarin Oral

26 Gall stone dissolution Chenodeoxycholic acid Chenix

27 Gout remedy Allopurinol Zyloprim

28 Immunosuppressant Cyclosporine Sandimmune

29 Tacrolimus (FK506) Prograf

30 Azathioprine Imuran

31 Lipd-lowering agent Probucol Lorelco

32 Gemfibrozil Lopid

33 Lovastatin Mevacor

34 Simvastatin Zocor

35 Fluvastatin Lescol

36 Niacin (nicotinic acid) Nicolar

37 Pravastatin sodium Pravachol

38 Nicotine delivery system Nicotine (transdermal) Nicoderm

39 NSAID Diclofenac Voltaren

40 Oxaprozin Daypro

41 Piroxicam Feldene

42 Naproxen Aleve

43 Psychoactive Alprazolam Xanax

44 Diazepam Valium

45 Fluoxetine Hydrochloride Prozac

46 Triazolam Halcion

47 Rheumatoid arthritis disease-modifier

Hydroxychloroquine Plaquenil

48 Sulfasalazine Azulfidine

49 Penicillamine Cuprimine

50 Skeletal muscle relaxants Dantrolene Dantrium

51 Thyroid replacement Levothyroxine Sodium Synthroid

Molecular Effects of Drugs™ (MED™) Database

LSP conducts an ongoing program examining the mechanisms

(therapeutic and toxic) of marketed pharmaceuticals, of which >50 have been studied

so far

Part of LSP's Rodent Molecular Effects Database

Successful Preclinical Marker Studies

• Monitor Therapeutic vs Toxicity Mechanisms• Cholesterol-lowering agents

• Compare Compound Mechanisms via SAR • PPAR nuclear receptor

• Recognize Mechanism Similarity Across Classes • 1,3-DT-2-T & Piroxicam

• Relate Toxic & Therapeutic Mechanisms • Cyclosporine Nephrotoxicity

• Detect Covalent Protein Adducts • Methapyrilene

• Detect Protein Phosphorylation• APAP vs AMAP Nuclear Effect

These studies are described in LSP scientific publications

Cholesterol Metabolism HMG CoA Reductase Inhibitors: Therapeutic vs toxicity mechanisms

• A tightly regulated pathway affecting a relatively small number of proteins

• Upregulated by lovastatin via inhibition of HMG CoA reductase

• Upregulated by cholestyramine via sequestration of bile acids

• Downregulated by high cholesterol diet (5%)

High Cholesterol (5%)

Control

Cholestyramine (1%)

Lovastatin (0.075%)

Lovastatin + Cholestyramine

Master 5 animals/group

Regulation of Liver Cytosolic HMG-CoA Synthase by Treatments Affecting Blood Cholesterol

21 Protein Spots AreAffected Through theCholesterol Pathway

6 Low Abundance Spots Are Strongly Induced

Cytosolic HMG CoA Synthase

Mitochondrial HMG CoA Synthase (fragment)

23kd Morphine- binding Protein (104)

PP strong- responder (367)

Effects of Cholesterol-Lowering Drugs in Rat Liver

Peroxisome Proliferators: SAR on PPAR

• Bind to PPAR Nuclear Receptors

• Trigger Induction of Fatty Acid Metabolism (etc? )

• Occur in Numerous Disparate Structural Classes

• Produce Liver Tumors in Rats

• No Apparent In Vivo Genotoxicity

A collaboration with Eli Lilly

OC

HO CH2

H2C

CH3

CH2

H2C

CH2

CH2

C

HN

N

N

N

O

H3C

C

HO CH2

H2C

H2C

CH3

O

H3C OH2C

C

HN

N

N

N

NH

N

N

H3C CH3

Cl

S CH2

C

O

O

WY14643

LY171883

LY163443*

-

C

O

O

H2C

CH

H2C

CH2

H2C

CH3

CH2

H3CC

O

O

CH2

CH

CH2

H2C

CH2

CH3

CH2

H3C

OCl

O

C

C

H3C CH3

O

O

C

C

H3C CH3

O

O

DEHP

Clofibric Acid

Nafenopin

-

-

Peroxisome Proliferators and Related Compounds*

SAR Comparison of Drug Effects by Quantitative Proteomics

The effects of peroxisome proliferators on protein abundances in mouse liver. Anderson, N.L., Esquer-Blasco, R., Richardson, F., Foxworthy, P. and Eacho, P. Toxicology and Applied Pharmacology, 137, 75-89, 1996.

OneCompound

200150

10090

8070

60

50

40

30

20

15

4.5 5.0 5.5 6.0 6.5 7.0

Cyt. b5

Actin

CPS

163

Albumin

HSP-60

MUP

CA-IIISMP-30

Calreticulin

SOD

Effects of Peroxisome Proliferators in Mouse LiverNafenopinWY14,643LY171,883LY163,443DEHPClofibrate

>10 1.25

1

2

0.25<10 0.5

0.8

4

P <0.000001P <0.00001

P <0.001P <0.0001

HSP-90

NP450R



200150

10090

8070

60

50

40

30

20

15

4.5 5.0 5.5 6.0 6.5 7.0

Actin

CPS

163

Albumin

HSP-60

MUP

CA-IIISMP-30

Calreticulin

SOD


>10 1.25

1

2

0.25<10 0.5

0.8

4

P <0.000001P <0.00001

P <0.001P <0.0001

HSP-90

NP450R

TwoCompounds



SixCompounds

200150

10090

8070

60

50

40

30

20

15

4.5 5.0 5.5 6.0 6.5 7.0

Actin

CPS

163

Albumin

HSP-60

MUP

CA-IIISMP-30

Calreticulin

SOD


>101.25

1

2

0.25<10 0.5

0.8

4

P <0.000001P <0.00001

P <0.001P <0.0001

HSP-90

NP450R

A= ControlB= LY163,443C= LY171,883 D= DEHPE= Clofibric acidF= WY14,643G= Nafenopin

Peroxisome Prolferators: 6 Compounds Compared Over 107 Selected Protein SpotsThe effects of peroxisome proliferators on protein abundances in mouse liver. Anderson, N.L., Esquer-Blasco, R., Richardson, F., Foxworthy, P. and Eacho, P. Toxicology and Applied Pharmacology, 137, 75-89, 1996.

AAA

AAA

A

A

A

AA

A

A A

A

B

B

B

BB

B

C

C

C

C

CCD

DD

DD

DE

EE E

EE

FF

FF

F

F GG

GG

G

G

Peroxisome proliferation response

LY163,443 response

Drug Mechanisms via Quantitative Proteomics:Patterns of Change in >100 Proteins Can Be Evaluated

to Reveal Mechanistic Similarities and Differences

Each symbol representsthe liver protein patternof an individual mouse

Dithiolethiones: Phase II enzyme induction, mechanism similarity across classes

SS

N

H3C

SOltipraz (parent)

SS

S

O

H3CAnethole Trithione

SS

S

1,2-Dithiole-3-thione

S

SS

1,3-Dithiole-2-thione

OC

CC

CO

CH3

O

H3C

O

Dimethyl fumarate

A collaboration with Division of Cancer Prevention and Control, Chemopreventive Investigational Drug Unit, US National Cancer Institute

o2 Group 15: Oltipraz (control)

O2 Group 16: Oltipraz (442umol)

a Group 17: ANTT (control)

A Group 18: ANTT (442umol)

m Group 19: DMF (control)

M Group 20: DMF (442umol)

r Group 21: 1,3-DT-2-T (control)

R Group 22: 1,3-DT-2-T (442umol)

w Group 23: 1,2-DT-3T (control)

W Group 24: 1,2-DT-3-T (442umol)

o2

o2

o2

o2

o2a a

aa a

m

m

m

mm

r

r

r

r r r

ww

w

wwO2

O2O2

O2

O2AA

AAA

M

M

MM

R

R

RR

R

W

W

W

W

-4 -3 -2 -1 0 1 2

FACTOR2: Potency as Phase II Enzyme Inducer

-2

-1

0

1

2

3

4

FA

CT

OR

1: P

oten

cy W

ith

Res

pect

to M

echa

nism

“R

”

Effects of Chemoprevention Compounds in Rat Liver: Two Mechanisms Are Detected Among the Dithiolethiones

Principal Components Analysis of Data on 81 Spots

Controls

Each symbol is an individualanimal’s protein expression pattern

o1 Group 1: Oltipraz 21 days in feed + 2 days recovery controls

i Group 2: Ibuprofen 21day feed control

f Group 3: DFMO 21day feed control

h Group 4: 4-HPR 21day feed control

cp Group 5: Carbenoxolone and Piroxicam 21day feed control

gd Group 6: Ca glucarate and DHEA analogue 21day feed cntrl

O1 Group 7: Oltipraz 21 days in feed + 2 days recvry 100mg/kg

I Group 8: Ibuprofen 21day feed MTD

F Group 9: DFMO 21day feed MTD

H Group 10: 4-HPR 21day feed MTD

C Group 11: Carbenoxolone 21 day feed MTD

P Group 12: Piroxicam 21 day feed MTD

G Group 13: Ca glucarate 21day feed MTD

D Group 14: DHEA analogue 21day feed MTD

o2 Group 15: Oltipraz (control)

O2 Group 16: Oltipraz (442umol)

a Group 17: ANTT (control)

A Group 18: ANTT (442umol)

m Group 19: DMF (control)

M Group 20: DMF (442umol)

r Group 21: 1,3-DT-2-T (control)

R Group 22: 1,3-DT-2-T (442umol)

w Group 23: 1,2-DT-3T (control)

W Group 24: 1,2-DT-3-T (442umol)

o1o1

o1o1o1

o1o1

o1

i

iii i

f

f

f

f

f

h h

hh

h

hcp cp

cp

cp cp

gd

gd

gd

gd

gd

O1O1

O1 O1O1

II

II

I

I

I

I

I

FF

F

F

F

FF

F

H

HH H

HH

H

H HHCCC

C

C

C

P

P

P

P

PP

P

G

G

GGGG

GGGG DD

DD

D

D

o2

o2

o2

o2

o2a a

aa a

m

m

m

mm

rr

r

r r r

ww

w

wwO2O2O2

O2

O2AA

AAA

M

MM

M

R

R

RR

R

W

W

W

W

-4 -3 -2 -1 0 1 2

-3

-2

-1

0

1

2

3

4

FACTOR2: Potency as Phase II Enzyme Inducer

FA

CT

OR

1: P

oten

cy W

ith

Res

pect

to M

echa

nism

“R

”

Effects of Chemoprevention Compounds in Rat Liver:Mechanism “R” Is Shared With Piroxicam

Principal Components Analysis of Data on 81 Spots

Cyclosporin:Markers of Mechanism-Specific Toxicity

• Potent immunosuppressive drug

• Used to prevent organ graft rejection

• Produces dose-limiting adverse side effects in kidney (tubular toxicity, kidney calcification, renal dysfunction)

• Pathogenesis unclear

A collaboration with Novartis Pharma

Kidney Protein Effects of CsA:Major Downregulation of Spot 75

CsA 15mg/kg/d 12days

CsA 50mg/kg/d 12 days

Arrow Plot here!

Mitochondrial:

67 Cytochrome C oxidase

peptide II

Cytosolic:

75 Calbindin 28kD

96 Senescence marker

protein 30

109 a-2U-globulin

119 Tropomyosin

6775

96

109

119

Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney. Steiner, S., Aicher, L., Cordier, A., Raymackers, J., Meheus, L., Esquer-Blasco, R., and Anderson, N.L. Biochem. Pharmacol., 51, 253-258, 1996.

Control kidney immuno-stained for calbindin d28

CsA-treated kidney immuno-stained for calbindin d28, showing calcium deposits

Histological Effects of CsA Treatment in Rat Kidney

Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney. Steiner, S., Aicher, L., Cordier, A., Raymackers, J., Meheus, L., Esquer-Blasco, R., and Anderson, N.L. Biochem. Pharmacol., 51, 253-258, 1996.

0

20

40

60

80

100

120

140

160

180

200

Control CsA FK-506 Rapamycin PSC-833

% o

f C

ontr

ol r

enal

cal

bind

in

10 days31 daysCsA, SDZ PSC 833: 50mg/kg/day

FK-506, Rapamycin: 5mg/kg/day

Drug-Effects on Renal Calbindin Levels

Methapyrilene:

• Histamine H1 receptor antagonist• Formerly used in many OTC and prescription

medications• Produces liver tumors in rats• No apparent In vivo genotoxicity• Causes proliferation of mitochondria

H2C

N

H2C

CH2

N

CH 3

N

CH 3

S

Methapyrilene H2C

N

H2C

CH2

N

CH 3

NO

CH3

Pyrilamine

Non-genotoxic liver carcinogen, novel covalent protein modifications

A collaboration with Eli Lilly

Methapyrilene Treatment Results in Charge Modification of Four Major Mitochondrial Proteins

1,000ppm MPYControl

grp75

HSP60

F1 ATPase beta

Carbamyl phosphate synthase

Protein Modification Correlates with Tumorigenicity

for Methapyrilene

Charge Modification

In vivo

In vitro

In vivo

Rat

-0.96

-0.81

Mouse

-0.14

-0.33

Human

-0.11Index

+

1 week

48 hr

Mitochondrial ProteinNA

Tumorigenicity

Strengths of Quantitative Proteomics

• Informative protein markers have been found for:– Disease states– Treatment effects (e.g., drugs)

• Therapeutic• Toxic

• Global analysis of marker patterns has:– Classified mechanisms and disease states– Allowed more sensitive detection than obtained with

single markers

pharmaceutical proteomics non-clinical studies subcommittee of the advisory committee for...

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