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1 Protein-protein interactions in cancer and Small molecule inhibitors of protein-protein interaction IPAM seminar April 26, 2004 Fuyu Tamanoi Juran Kato-Stankiewicz 2 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 3 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 4 5 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 6 PDZ-RhoGEF integrates G-protein coupled receptor signaling and plexin signaling Pawson and Nash (2003) Science 300, 445 7 Pawson et al (2001) Trends in Cell Biol 11, 504 8 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 9 The p53 Signaling Pathway 10 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 11 12 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 13 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 14 15 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, 16 17 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 18 Mutations of the ras oncogene are associated with a wide range of human cancers 19 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 84 33 44 53 35 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 20 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 21 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 22 The yeast two-hybrid assay to identify inhibitors of Ras/Raf interaction SKY54 Ras-Raf SKY54 hsRPB7hsRPB4 23 Putative Ras-Raf inhibitor MCP Putative antifungals Kato-Stankiewicz et al (2002) PNAS 99, 14398 24 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 25 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 140 120 100 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 26 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 27 28 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 29 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 30 31 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 32 33 “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: 34 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 35 36 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.