1. Design and synthesis of novel Grb2 SH2 domain binding antagonists. In many human cancers, dysregulated growth factor receptor tyrosine kinase (RTK) activation contributes to tumor cell survival, proliferation and invasiveness, as well as tumor angiogenesis. These events are critical for cancer progression and thus logical targets for therapeutic intervention. RTK activation leads to autophosphorylation of specific tyrosine residues within RTK intracellular domains and the consequent formation of docking sites for proteins containing SH2 domains. One of the best characterized proteins of this class is Grb2, an adapter protein that acts as a critical downstream intermediary in several oncogenic signaling pathways. Originally isolated through screening for epidermal growth factor receptor (EGFR) interacting proteins, Grb2 associates with several other signaling and regulatory proteins. Through its SH2 domain, which is a conserved sequence of approximately 100 amino acids, Grb2 can interact directly with RTKs and non-receptor tyrosine kinases (e.g. focal adhesion kinase (FAK) and Bcr/Abl) by preferential binding to phosphopeptide motifs of the form pYXNX, where pY represents phosphotyrosine, N is asparagine and X is any residue. The amino- and carboxyl-terminal Src homology 3 (SH3) domains of Grb2, which have a conserved sequence of around 50 amino acids, bind proline-rich regions within additional interacting proteins. Through these two SH3 domains, Grb2 links activated RTKs with several key intracellular regulatory networks, including the Ras/Erk pathway controlling cell cycle progression, and the p21-activating kinase (PAK1) and Arp2/3/WASp pathways regulating the actin filament system, cell shape change and motility. Grb2 is also a key intermediate of integrin signaling through its interaction with activated FAK at pY925, which resides within the canonical recognition motif for the Grb2 SH2 domain. Grb2-FAK binding triggers a signaling sequence involved in angiogenesis and epithelial-mesenchymal transition (EMT), both of which are important contributors to tumor progression. The critical roles served by Grb2 in cell motility and angiogenesis make it a logical therapeutic target for pathological processes leading to the spread of solid tumors through local invasion and metastasis. Based on the unique three-dimensional structure of the Grb2 SH2 domain and its unique amino acid recognition sequence, we designed and synthesized potent, synthetic, low molecular weight antagonists of Grb2 SH2 domain binding. Since the cytosolic environment has a high level of constitutive tyrosine phosphatase activity, stabilizing this moiety in vivo was a major challenge. We initially achieved phosphatase resistance by replacing the pY residue with phosphonomethyl phenylalanine, and we were the first to develop active compounds using non-phosphate containing groups. Systematic substitution of the pYXN recognition motif led to high affinity synthetic compounds that blocked RTK-Grb2 interactions in intact cells. Macrocyclization was later used to stabilize the required β-turn conformation, further improving potency. 2. Defining the roles of Grb2 in oncogenesis and metastasis. We have shown that the Grb2 SH2 domain binding antagonists described here potently block RTK-Grb2 interaction and growth factor-stimulated motility and matrix invasion by several tumor cell lines, including those derived from RCC, prostate cancer, bladder cancer, leimyosarcoma, gastric carcinoma and glioblastoma. We have also found that hepatocyte growh fator (HGF)-induced Met-Grb2 binding and Grb2/FAK interaction were significantly disrupted by these agents in intact cells, resulting in dramatically reduced focal adhesion and lamellipodia formation in an aggressive human prostate cancer cell line. These results defined a critical role for Grb2 in processes that are known to mediate growth factor-stimulated cell motility. The same compounds also inhibit the basic morphogenetic events required for angiogenesis, such as growth factor-driven endothelial cell migration and matrix invasion. Moreover, inhibition of angiogenesis in an in vitro by human endothelial cells, and suppression of vasculogenesis in the chick chorioallantoic assay, further implicate Grb2 in pro-angiogenic pathways and reinforce the concept that its blockade is a viable anti-angiogenesis strategy. 3. Preclinical development of SH2 domain antagonists as anti-cancer drugs. Because Grb2 SH2 domain binding antagonists were found to be highly effective in blocking cell motility, models of tumor metastasis were sought for the preclinical development of these compounds in animals. Using a murine melanoma cell line in an experimental metastasis model and a human prostate adenocarcinoma cell line in a spontaneous metastasis model, in conjunction with bioluminescence technology to quantitate metastasis, a significant reduction in metastatic burden was achieved using a prototypical Grb2 SH2 domain binding antagonist. These results demonstrated that it was possible to specifically target the spread of solid tumors using small molecules and it implied a critical role for the Grb2 SH2 domain in that process. Several important steps remain in the preclinical development of Grb2 SH2 domain binding antagonists as anti-cancer drugs. These compounds are among the first and most successful antagonists of protein-protein interactions, but defining target selectivity and the molecular basis of therapeutic effects are important steps toward their development and use. This project is now co-sponsored by Developmental Therapeutics Program (DTP) in the NCI Division of Cancer treatment and Diagnosis, which provides extensive resources for the study of drug formulation, pharmacokinetics, toxicology and drug efficacy. In addition, to better understand SH2 domain selectivity and mechanism of action, we developed biotinylated derivatives of two biologically active structures and established that high affinity Grb2 binding was fully retained. Selectivity was then investigated ligand via target protein immobilization with streptavidin-coated beads and mass spectrometry (MS) analysis. Importantly, Grb2 was the only SH2-domain containing protein detected by MS among all proteins captured from cell lysates. These results confirmed and extended our prior findings that these structures selectively block the Grb2 SH2 domain and not other, structurally distinct SH2 domains. Initial pharmacokinetic studies of a Grb2 SH2 domain binding antagonist in mice indicate that while it had a relative short biological half-life, target drug doses could be maintained continuously using implanted osmotic minipumps. Further studies of drug formulation in lipophilic nanoparticles to improve half-life and bioavailability are also underway in collaboration with DTP. Thus far, no toxicity has been observed in mice treated for four weeks with doses designed to exceed the ED50 by 100-fold, and further toxicological studies are in progress. Finally, the development of pharmacodynamic markers of drug action in intact animals represents another high priority in the preclinical development of our Grb2 SH2 domain antagonists. In collabortaion with DTP, global analysis of gene expression changes associated with drug treatment with will be used to identify a molecular signature of drug effect;preliminary studies suggest that N-cadherin, implicated in the increased invasiveness of many cancers, is among this set of potential pharmacodynamic markers.
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