Title: Protein-protein interactions in cancer
11
Protein-protein interactions in cancer and Small
molecule inhibitors of protein-protein interaction
IPAM seminar April 26, 2004
Fuyu Tamanoi Juran Kato-Stankiewicz
22
Signal Transduction and Cancer
Signal Tranduction
Apoptosis
Differentiation
Proliferation
Gene expression Cell cycle
Loss of tumor suppressors Oncogenes
Aberrant signal transduction
3The Growth factor signaling pathway
3
SIGMA-ALDRICH
44
Protein-protein interaction in signal transduction
- Protein-protein interaction as adaptors and
signal integrators - Modular binding domain
- SH2, SH3 domains
- PDZ domains
- Protein-protein interaction as inhibitors of
protein function - Caspase inhibitors Caspase/IAP
- p53 inhibitors p53/Mdm2
- Protein-protein interaction as activators of
protein function - G-protein/protein kinase interaction
- Ras/Raf kinase
- Rho/Rho kinase
- Rac/Pak kinase
55
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
66
PDZ-RhoGEF integrates G-protein coupled receptor
signaling and plexin signaling
Pawson and Nash (2003) Science 300, 445
77
Pawson et al (2001) Trends in Cell Biol 11, 504
88
Assembly of Cell Regulatory Systems through
Protein Interaction Domains
Pawson and Nach (2003) Science 300, 445
9Modulation of signal transduction by
disrupting modular domain interactions
9
Peptidomimetic inhibitors of SH2/pY interaction
Sundaramoorthi et al (2003) Biopolymers 71, 717
10The p53 Signaling Pathway
10
SIGMA-ALDRICH
1111
p53 tumor suppressor is downregulated by 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
Mdm2
The mdm2 gene has been found amplified or
overexpressed in many human malignancies.
p53
Mdm2
X
Activation and increase of p53
Cell cycle arrest Apoptosis Inhibition of tumor
growth
1212
Nutlin-2 fits in the p53 binding pocket of Mdm2
Structure of the p53-Mdm2 complex
Vassilev et al (2004) Science 303, 844
1313
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
14Programmed Cell Death
14
SIGMA-ALDRICH
IAP
1515
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.
16Caspase/XIAP inhibitor, TWX compounds, bind to
the BIR2 domain of XIAP.
16
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,
1717
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
18Ras plays critical roles in the signaling
pathway leading to transformation
18
RTK
GRB2
SOS1,2
GAP NF1
Ras GTP
Ras GDP
Raf -1, A-, B-
RalGEFs
PI3K
Ral
MEK1,2
AKT
ERK1,2
Survival
Transcription Factors
Muegge et al (1996) Structure 4, 475
1919
Mutations of the ras oncogene are associated with
a wide range of human cancers
The ras oncogene causes oncogenic transformation.
Mutations in the ras oncogene are found in a wide
range of human cancer
Number of samples with a
ras gene Tumor samples tested mutated
ras gene found to be mutated
Pancreas adenocarcinoma 156 84 K Lung
adenocarcinoma 45
33 K Colon adenocarcinoma 277
44 K Thyroid follicular carcinoma 15
53 H, K, N Myeloid disorder
(AML) 412 35
N, K
Bos, JL (1989) Cancer Res. 49, 4682
These mutations lead to constitutive activation
of the Ras signaling pathway.
2020
Constitutive activation of Ras
GEF
F
F
GTP
GDP
Ras
Ras
Pi
GTP
GDP
Effectors Raf, PI3K, RalGDS
GAP
Ras mutations inhibit GTPase activity,
causing constitutive activation of Ras
2121
Ras
GTP
Muegge et al (1996) Structure 4, 475
RapRaf-RBD interface
Bax and Jhoti (1995) Curr. Biol. 5, 1119
2222
Yeast two-hybrid based screen for the inhibitors
of Ras-Raf interaction
Raf-1
H-Ras
Blue colony
AD
cI
Raf-1
White colony
H-Ras
X
AD
cI
hsRPB4
hsRPB7
Blue colony
X
AD
23The yeast two-hybrid assay to identify inhibitors
of Ras/Raf interaction
23
Putative Ras-Raf inhibitor
MCP
SKY54
Ras-Raf
Putative antifungals
Putative antifungals
SKY54
hsRPB7-
hsRPB4
Kato-Stankiewicz et al (2002) PNAS 99, 14398
2424
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)
25MCP, novel Ras/Raf inhibitors
25
MCP1
C29H27ClN2O3 MW 487
MCP110
MCP122
C33H36N2O3
C22H24N2O2
Decreased Activity
Enhanced activity
26MCP inhibits Raf/MEK/ERK activation in HT-1080
cells
MCP
26
Kato-Stankiewicz et al (2002) PNAS 99, 14398
27MCP induces G1 arrest of a lung cancer cell line
A549
Ras
Ras
MCP
Raf
Raf
MEK
MEK
MAPK
MAPK
p27
p27
CyclinD
CyclinD
CDK4/6
CDK4/6
CyclinE
CyclinE
s
e
r
u
m
s
e
r
u
m
E
G
F
E
G
F
P
D
G
F
P
D
G
F
CDK2
CDK2
Cell cycle
Cell cycle
MCP110
MCP110
Cyclin
D 1/2
Cyclin
D 1/2
Kato-Stankiewicz et al (2002) PNAS 99, 14398
27
2828
MCP inhibits anchorage-independent growth of
human cancer cells
MCP1
DMSO
Fibrosarcoma
N-ras (Q61K)
HT-1080
Lung cancer
K-ras (G12S)
A549
Pancreatic cancer
K-ras (G12V)
PANC-1
Melanoma
B-raf (V599E)
A2058
Kato-Stankiewicz et al (2002) PNAS 99, 14398
2929
MCP induces flat reversion of H-ras(G12V)
transformed NIH3T3 cells
Kato-Stankiewicz et al (2002) PNAS 99, 14398
3030
MCP reverses Ras-transformed phenotypes of human
cancer cells
Ras transformation
Inhibition of apoptosis
Morphological changes and loss of actin stress
fibers
Metastasis Invasive properties Motility VEGF and
angiogenesis
Anchorage- independent growth
Cell cycle change
Ras transformed phenotypes
3131
Small molecule inhibitors of the Ras signaling
pathway
FTI
RTK
GRB2
SOS1,2
GAP NF1
Ras GTP
Ras GDP
ZD1839 (Iressa)
MCP
Raf -1, A-, B-
BAY43-9006
RalGEFs
PI3K
AKT
Ral
MEK1,2
CI-1040
Survival
ERK1,2
Transcription Factors
32Small molecule inhibitors of protein-protein
interactions
32
Compound Target Phenotype Receptor/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 c
almodulin/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
3333
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
3434
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.
35Protein-protein interaction assays
35
- Biochemical assays
- co-IP, GST-pull down, ELISA,
- Tandem affinity purification
- 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
3636
Small molecule inhibitors of protein-protein
interaction
- Powerful means to modulate signal transduction
pathways. - Potential as anti-cancer drugs.
- Chemical compound database.
- Protein-protein interaction interface.