Transcript No Slide Title

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Protein-protein interactions in cancer
and
Small molecule inhibitors of protein-protein interaction
IPAM seminar
April 26, 2004
Fuyu Tamanoi
Juran Kato-Stankiewicz
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Signal Transduction and Cancer
Signal Tranduction
Apoptosis
Differentiation
Proliferation
Gene expression
Cell cycle
Loss of tumor suppressors
Oncogenes
Aberrant signal transduction
The Growth factor signaling pathway
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SIGMA-ALDRICH
Protein-protein interaction in signal transduction
1. Protein-protein interaction as adaptors and signal integrators
Modular binding domain
SH2, SH3 domains
PDZ domains
2. Protein-protein interaction as inhibitors of protein function
Caspase inhibitors
Caspase/IAP
p53 inhibitors
p53/Mdm2
3. Protein-protein interaction as activators of protein function
G-protein/protein kinase interaction
Ras/Raf kinase
Rho/Rho kinase
Rac/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 signaling
and 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
Modulation of signal transduction by disrupting
modular domain interactions
Peptidomimetic inhibitors
of SH2/pY interaction
Sundaramoorthi et al
(2003) Biopolymers 71, 717
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The p53 Signaling Pathway
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SIGMA-ALDRICH
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.
The mdm2 gene has been found amplified or overexpressed in many
human malignancies.
p53
X
Mdm2
Activation and increase of p53
Cell cycle arrest
Apoptosis
Inhibition of tumor growth
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Structure of the p53-Mdm2 complex
Three p53 residues (Phe19, Trp23
and Leu26) contribute to a large
extent to the interaction.
Chene et al (2003) Nat Rev. Cancer3, 102
Nutlin-2 fits in the p53 binding pocket
of Mdm2
Vassilev et al (2004) Science 303, 844
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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 systems
Human tumor xenografts
Vassilev et al (2004) Science 303, 844
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.
Caspase/XIAP inhibitor, TWX compounds, bind to
the BIR2 domain of XIAP.
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,
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Caspase/XIAP inhibitors
TWX compounds, Polyphenyl urea comp.
Induces apoptosis and sensitizes cancer cells
to chemotherapeutic drugs
Wu et al (2003) Chem. & Biol.10, 759
Schimmer et al (2004) Cancer Cell 5, 25
Ras plays critical roles in the signaling pathway
leading to transformation
RTK
GRB2 SOS1,2
Ras
GTP
GAP
NF1
Raf
-1, A-, B-
PI3K RalGEFs
MEK1,2
AKT
ERK1,2
Muegge et al (1996)
Structure 4, 475
Ras
GDP
Transcription
Factors
Survival
Ral
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Mutations of the ras oncogene are associated with a
wide range of human cancers
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The ras oncogene causes oncogenic transformation.
Mutations in the ras oncogene are found in a wide range of human
cancer
Tumor
Number of
samples tested
Pancreas adenocarcinoma
Lung adenocarcinoma
Colon adenocarcinoma
Thyroid follicular carcinoma
Myeloid disorder (AML)
156
45
277
15
412
% samples with a
mutated ras gene
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44
53
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ras gene
found to be
mutated
K
K
K
H, K, N
N, K
Bos, JL (1989) Cancer Res. 49, 4682
These mutations lead to constitutive activation of the Ras signaling pathway.
Constitutive activation of Ras
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GEF
F
F
GTP GDP
Ras
Ras
GDP
Pi
GTP
GAP
Effectors
Raf, PI3K,
RalGDS
Ras mutations inhibit GTPase activity, causing
constitutive activation of Ras
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Ras GTP
Muegge et al (1996)
Structure 4, 475
Effector binding domain
(aa 32-40)
Rap–Raf-RBD interface
Bax and Jhoti (1995) Curr. Biol. 5, 1119
Yeast two-hybrid based screen for the inhibitors of
Ras-Raf interaction
Raf-1
H-Ras
Blue colony
cI
AD
Raf-1
H-Ras
White colony
X
cI
AD
hsRPB4
hsRPB7
Blue colony
AD
X
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The yeast two-hybrid assay to identify inhibitors
of Ras/Raf interaction
SKY54
Ras-Raf
SKY54
hsRPB7hsRPB4
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Putative Ras-Raf inhibitor
MCP
Putative antifungals
Kato-Stankiewicz et al (2002) PNAS 99, 14398
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High throughput screen
73,400 chemical compound library
High throughput yeast two-hybrid assay
38 compounds
c-fos-sre-Luciferase assay (Mammalian cell based)
13 compounds
MCP compounds
In collaboration with Morphochem (Khazak/Golemis/Weber)
MCP, novel Ras/Raf inhibitors
MCP1
C29H27ClN2O3
MW 487
MCP110
MCP122
C33H36N2O3
C22H24N2O2
Decreased
Activity
Enhanced
activity
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MCP
Raf
100
80
60
40
20
0
(mM)
MCP
Raf-1
- 20 1 2 5 10 20
- 122
110
MEK-1 activity (%)
Ras
Raf-1 activity (%)
MCP inhibits Raf/MEK/ERK activation in HT-1080 cells
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120
100
80
60
40
20
0
MEK-1
-
122 110 PD U0126
MEK
ERK
IB: phospho-ERK1/2
IB: ERK1/2
ERK1/2
Kato-Stankiewicz et al (2002) PNAS 99, 14398
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MCP induces G1 arrest of a lung cancer cell line A549
100
Cell number (%)
Ras
MCP
Raf
MEK
MAPK
p27
CyclinD
CDK4/6
CyclinE
CDK2
Cell cycle
80
G1
S
G2/M
60
40
20
0
-
MCP122 MCP110
E G F PDGF se ru m
+
+
+
MCP110
Cyclin D 1/2
Kato-Stankiewicz et al (2002) PNAS 99, 14398
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MCP inhibits anchorage-independent growth of human cancer cells
DMSO MCP1
HT-1080
Fibrosarcoma
N-ras (Q61K)
A549
Lung cancer
K-ras (G12S)
PANC-1
Pancreatic cancer K-ras (G12V)
A2058
Melanoma
B-raf (V599E)
Kato-Stankiewicz et al (2002) PNAS 99, 14398
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MCP induces flat reversion of
H-ras(G12V) transformed NIH3T3 cells
2 mM
5 mM
10 mM
MCP53
DMSO
0.1 %
Kato-Stankiewicz et al (2002) PNAS 99, 14398
MCP reverses Ras-transformed phenotypes
of human cancer cells
Ras transformation
Morphological
changes and loss
of actin stress fibers
Cell cycle
change
Inhibition of
apoptosis
Anchorageindependent
growth
Metastasis
Invasive properties
Motility
VEGF and angiogenesis
Ras transformed phenotypes
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Small molecule inhibitors of the Ras signaling pathway
FTI
RTK
GRB2 SOS1,2
ZD1839
(Iressa)
MCP
BAY43-9006
CI-1040
Ras
GTP
GAP
NF1
Raf
-1, A-, B-
MEK1,2
ERK1,2
Transcription
Factors
Ras
GDP
PI3K RalGEFs
AKT
Survival
Ral
Small molecule inhibitors of protein-protein interactions
Compound
Target
Receptor/agonist
TSR1265
Integrinavb3/MMP
ALE0540
TrkA/NGF
TAK779
CCR5/RANTES/HIV
Cyclic peptide
C5aR/agonist(s)
Cytosolic signaling molecules
UCS15A
SrcSH3/Sam68
AP22161/AP22408
Lck/Src-SH2/pY
Trifluoroperazine
calmodulin/ATPase
BH32, HA14-1
Bcl-2 family heterodimers
Nutlins
p53/mdm2
TWX
Caspase/XIAP
Geldanamycin
Hsp90/p23 co-Chaperone
MCP
Ras/Raf
Transcription factors
IIA4B11
Myc/Max
Phenotype
Abolishes angiogenesis
Reduces neutropenia
Reverts v-src- transformation
Inhibit bone resorption
Induce apoptosis
Cell cycle arrest and apoptosis
Induce apoptosis
Reverts ras-transformation
Inhibits growth of
Myc-transformed fibroblasts
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“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
Flat and quite large interface
Hot spots representing energetic focal points exist
Changes in our thinking of the nature of protein interfaces:
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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-2Ra binding hot spots of IL-2 and induces
changes in the conformation of IL-2 protein.
Ro26-4550
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.
Protein-protein interaction assays
1. Biochemical assays
co-IP, GST-pull down, ELISA,
Tandem affinity purification
2. Biophysical assays
Fluorescent resonance energy transfer (FRET) assay
Surface plasmon resonance using Biacore
Isothermal calorimetric analysis
Atomic force microscopy
Quartz 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.