Upload
ambrose-barnett
View
219
Download
1
Tags:
Embed Size (px)
Citation preview
Protein-protein interactions in cancerand
Small molecule inhibitors of protein-protein interaction
IPAM seminarApril 26, 2004
Fuyu TamanoiJuran Kato-Stankiewicz
1
Signal Transduction and Cancer
Gene expressionCell cycle
Loss of tumor suppressorsOncogenes
Signal Tranduction
Proliferation
ApoptosisDifferentiation
Aberrant signal transduction
2
The Growth factor signaling pathway
SIGMA-ALDRICH
3
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
4
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
5
PDZ-RhoGEF integrates G-protein coupled receptor signalingand plexin signaling
Pawson and Nash (2003) Science 300, 445
6
Pawson et al (2001) Trends in Cell Biol 11, 504
7
Assembly of Cell Regulatory Systems through Protein Interaction Domains
Pawson and Nach (2003)Science 300, 445
8
Peptidomimetic inhibitors of SH2/pY interaction
Sundaramoorthi et al(2003) Biopolymers 71, 717
Modulation of signal transduction by disruptingmodular domain interactions
9
The p53 Signaling Pathway SIGMA-ALDRICH
10
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.
11
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
12
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
13
Programmed Cell Death SIGMA-ALDRICH
IAP
14
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.
15
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.
16
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
17
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
18
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.
19
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
20
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
21
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
22
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
23
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)
24
MCP, novel Ras/Raf inhibitors
MCP1
C29H27ClN2O3
MW 487
C33H36N2O3 C22H24N2O2
MCP110 MCP122
Enhanced activity
DecreasedActivity
25
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
28
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
29
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
30
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)
31
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
33
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
34
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
35
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.
36