Protein-protein interactions in cancerand

Small molecule inhibitors of protein-protein interaction

IPAM seminarApril 26, 2004

Fuyu TamanoiJuran Kato-Stankiewicz

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Signal Transduction and Cancer

Gene expressionCell cycle

Loss of tumor suppressorsOncogenes

Signal Tranduction

Proliferation

ApoptosisDifferentiation

Aberrant signal transduction

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The Growth factor signaling pathway

SIGMA-ALDRICH

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Protein-protein interaction in signal transduction

1. Protein-protein interaction as adaptors and signal integratorsModular binding domain

SH2, SH3 domainsPDZ domains

2. Protein-protein interaction as inhibitors of protein functionCaspase inhibitors Caspase/IAPp53 inhibitors p53/Mdm2

3. Protein-protein interaction as activators of protein functionG-protein/protein kinase interaction

Ras/Raf kinaseRho/Rho kinaseRac/Pak kinase

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Grb2 acts as an adaptor that links receptor activation and Ras activation as well as recruitment of PI3K

Pawson et al (2001) Trends in Cell Biol 11, 504

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PDZ-RhoGEF integrates G-protein coupled receptor signalingand plexin signaling

Pawson and Nash (2003) Science 300, 445

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Pawson et al (2001) Trends in Cell Biol 11, 504

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Assembly of Cell Regulatory Systems through Protein Interaction Domains

Pawson and Nach (2003)Science 300, 445

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Peptidomimetic inhibitors of SH2/pY interaction

Sundaramoorthi et al(2003) Biopolymers 71, 717

Modulation of signal transduction by disruptingmodular domain interactions

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The p53 Signaling Pathway SIGMA-ALDRICH

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p53 tumor suppressor is downregulated by Mdm2

p53 Mdm2

Blocks the ability of p53 to activate transcription.Serves as a ubiquitin ligase that promotes p53 degradation.Involved in the nuclear export of p53.

p53 Mdm2X Activation and increase of p53

Cell cycle arrestApoptosis Inhibition of tumor growth

The mdm2 gene has been found amplified or overexpressed in manyhuman malignancies.

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Structure of the p53-Mdm2 complexNutlin-2 fits in the p53 binding pocket

of Mdm2

Vassilev et al (2004) Science 303, 844

Three p53 residues (Phe19, Trp23and Leu26) contribute to a largeextent to the interaction.

Chene et al (2003) Nat Rev. Cancer3, 102

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Nutlins

Induces cell cycle arrest and apoptosis of human cancer cells

The effects seen only with p53 expressing cells.

Inhibits growth of tumors in mouse model systemsHuman tumor xenografts

Vassilev et al (2004) Science 303, 844

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Programmed Cell Death SIGMA-ALDRICH

IAP

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Caspase/IAP interaction

Caspase IAP

IAP family proteins are caspase inhibitors sharing a conserved structure BIR domain.

XIAP is the most potent suppressor of cell death.

XIAP levels are pathologically elevated in leukemia,prostate cancer and lung cancer.

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Chemistry & Biology Volume 10, Issue 8 , August 2003, Pages 759-767 Development and Characterization of Nonpeptidic Small Molecule Inhibitors of the XIAP/Caspase-3 Interaction

Tom Y. H. Wu1, Klaus W. Wagner2, Badry Bursulaya2, Peter G. Schultz1, 2, , and Quinn L. Deveraux2,

Caspase/XIAP inhibitor, TWX compounds, bind to the BIR2 domain of XIAP.

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Caspase/XIAP inhibitors

TWX compounds, Polyphenyl urea comp.

Induces apoptosis and sensitizes cancer cellsto chemotherapeutic drugs

Wu et al (2003) Chem. & Biol.10, 759Schimmer et al (2004) Cancer Cell 5, 25

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GRB2 SOS1,2Ras

GDP

RasGTP

RTK

Raf -1, A-, B-

MEK1,2

ERK1,2

TranscriptionFactors

GAPNF1

PI3K

AKT

Survival

RalGEFs

Ral

Ras plays critical roles in the signaling pathwayleading to transformation

Muegge et al (1996) Structure 4, 475

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Mutations in the ras oncogene are found in a wide range of human cancer

Number of % samples with a ras geneTumor samples tested mutated ras gene found to be

mutated

Pancreas adenocarcinoma 156 84 K Lung adenocarcinoma 45 33 KColon adenocarcinoma 277 44 KThyroid follicular carcinoma 15 53 H, K, NMyeloid disorder (AML) 412 35 N, K

Bos, JL (1989) Cancer Res. 49, 4682

These mutations lead to constitutive activation of the Ras signaling pathway.

Mutations of the ras oncogene are associated with a wide range of human cancers

The ras oncogene causes oncogenic transformation.

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Constitutive activation of Ras

F

GDP

F

GTPPi

GEF

GTP GDP

GAP

RasRas

Ras mutations inhibit GTPase activity, causingconstitutive activation of Ras

EffectorsRaf, PI3K,RalGDS

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Ras GTP

Effector binding domain (aa 32-40) Rap–Raf-RBD interface

Muegge et al (1996) Structure 4, 475

Bax and Jhoti (1995) Curr. Biol. 5, 1119

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AD

Blue colonyH-Ras Raf-1

cI

AD

H-Ras

Raf-1

cI

White colonyX

ADX

Blue colony

hsRPB4 hsRPB7

Yeast two-hybrid based screen for the inhibitors of Ras-Raf interaction

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SKY54

hsRPB4

Putative antifungals

SKY54Ras-Raf

hsRPB7-

Putative Ras-Raf inhibitorMCP

Putative antifungals

The yeast two-hybrid assay to identify inhibitors of Ras/Raf interaction

Kato-Stankiewicz et al (2002) PNAS 99, 14398

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High throughput screen

73,400 chemical compound library

38 compounds

13 compounds

c-fos-sre-Luciferase assay (Mammalian cell based)

MCP compounds

High throughput yeast two-hybrid assay

In collaboration with Morphochem (Khazak/Golemis/Weber)

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MCP, novel Ras/Raf inhibitors

MCP1

C29H27ClN2O3

MW 487

C33H36N2O3 C22H24N2O2

MCP110 MCP122

Enhanced activity

DecreasedActivity

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MCP inhibits Raf/MEK/ERK activation in HT-1080 cells

020406080

100

Raf

-1 a

ctiv

ity

(%)

- 20 1 2 5 10 20110122-

(M)MCP

Raf-1

020406080

100120140

- 122 110 PD U0126ME

K-1

act

ivit

y (%

)

MEK-1Ras

Raf

MEK

ERK

ERK1/2

- MCP12

2

MCP11

0

U0126

- MCP12

2

MCP11

0

U0126

IB: phospho-ERK1/2

IB: ERK1/2

MCP

Kato-Stankiewicz et al (2002) PNAS 99, 1439826

MCP induces G1 arrest of a lung cancer cell line A549

0

20

40

60

80

100

-MCP122 MCP110

G1S

G2/M

Cel

l num

ber

(%

)0

20

40

60

80

100

-MCP122 MCP110

G1S

G2/M

Cel

l num

ber

(%

)Ras

Raf

MAPK

CyclinD

CDK4/6

p27

CyclinECDK2

Cell cycle

MEK

MCP

Ras

Raf

MAPK

CyclinD

CDK4/6

p27

CyclinECDK2

Cell cycle

MEK

+ + + MCP110

E G F PDGF seru m

Cyclin D 1/2

+ + + MCP110

E G FE G F PDGFPDGF seru mseru m

Cyclin D 1/2

Kato-Stankiewicz et al (2002) PNAS 99, 1439827

Fibrosarcoma N-ras (Q61K)

Lung cancer K-ras (G12S)

Pancreatic cancer K-ras (G12V)

Melanoma

MCP inhibits anchorage-independent growth of human cancer cells

MCP1DMSO

HT-1080

A549

PANC-1

A2058 B-raf (V599E)

Kato-Stankiewicz et al (2002) PNAS 99, 14398

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2 M 5 M 10 MDMSO 0.1 %

MC

P53

MCP induces flat reversion of H-ras(G12V) transformed NIH3T3 cells

Kato-Stankiewicz et al (2002) PNAS 99, 14398

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Ras transformed phenotypes

Ras transformation

Morphologicalchanges and loss

of actin stress fibersAnchorage-independent

growth

MetastasisInvasive properties

MotilityVEGF and angiogenesisCell cycle

change

MCP reverses Ras-transformed phenotypes of human cancer cells

Inhibition of apoptosis

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Small molecule inhibitors of the Ras signaling pathway

GRB2 SOS1,2RasGDP

RasGTP

RTK

Raf -1, A-, B-

MEK1,2

ERK1,2

PI3K

AKT

Survival

TranscriptionFactors

GAPNF1

RalGEFs

Ral

FTI

MCPBAY43-9006

CI-1040

ZD1839(Iressa)

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Compound Target PhenotypeReceptor/agonist

TSR1265 Integrinavb3/MMP Abolishes angiogenesis

ALE0540 TrkA/NGF

TAK779 CCR5/RANTES/HIV

Cyclic peptide C5aR/agonist(s) Reduces neutropenia

Cytosolic signaling molecules

UCS15A SrcSH3/Sam68 Reverts v-src- transformation

AP22161/AP22408 Lck/Src-SH2/pY Inhibit bone resorption

Trifluoroperazine calmodulin/ATPase

BH32, HA14-1 Bcl-2 family heterodimers Induce apoptosis

Nutlins p53/mdm2 Cell cycle arrest and apoptosis

TWX Caspase/XIAP Induce apoptosis

Geldanamycin Hsp90/p23 co-Chaperone

MCP Ras/Raf Reverts ras-transformation

Transcription factors

IIA4B11 Myc/Max Inhibits growth of

Myc-transformed fibroblasts

Small molecule inhibitors of protein-protein interactions 32

“The disruption of protein-protein interactions represents one of the most challenging target classes for

small molecule drug discovery.”Thanos et al (2003) JACS 125, 15280

Changes in our thinking of the nature of protein interfaces:

Flat and quite large interface

Hot spots representing energetic focal points exist

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J Am Chem Soc. 2003 125(50):15280-1.

Potent small-molecule binding to a dynamic hot spot on IL-2.Thanos CD, Randal M, Wells JA.

Binding of small molecule compound induces conformational changes that facilitate binding to the target protein

A small molecule inhibitor of IL-2/IL-2 receptor, Ro26-4550, binds to the IL-2Rbinding hot spots of IL-2 and induceschanges in the conformation of IL-2 protein.

Ro26-4550

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Protein-protein interaction assays

1. Biochemical assaysco-IP, GST-pull down, ELISA, Tandem affinity purification

2. Biophysical assaysFluorescent resonance energytransfer (FRET) assay

Surface plasmon resonance using BiacoreIsothermal calorimetric analysisAtomic force microscopyQuartz crystal microbalance biosensor

3. The yeast two-hybrid assay

4. Luciferase complementation assay

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Small molecule inhibitors of protein-protein interaction

1. Powerful means to modulate signal transduction pathways.

2. Potential as anti-cancer drugs.

3. Chemical compound database.

4. Protein-protein interaction interface.

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