Aberrant kinase-depenent signaling is associated with the etiology of several cancers. For this reason, pharmacological agents are being developed to modulate kinase-dependent signaling as potential new anticancer therapeutics. Kinase-dependent signaling involves three critical components: (1) The generation of phosphorylated amino acid residues in key cellular proteins;(2) the recognition and binding to these residues by other signaling proteins and (3) the removal of the amino acid phosphoryl group by cellular phosphatases. For protein-tyrosine kinases (PTKs) these three components consist of: (1) The generation of phosphotyrosyl (pTyr)-containing sequences by PTKs;(2) The recognition and binding to pTyr-containing sequences by src homology 2 (SH2) domains and (3) The destruction of pTyr sequences through phosphate ester hydrolysis by protein tyrosine phosphatases (PTPs). Accordingly, a unifying theme of this project is the design and synthesis of inhibitors directed at each of these three components. In the SH2 domain area, high affinity growth factor receptor-bound protein 2 (Grb2)-binding antagonists are being prepared as potential new therapeutics for erbB-2 and c-Met dependent cancers. As part of a collaborative effort with NCI clinical investigators (Drs. Don Bottaro and Marston Linehan), our Grb2 signaling inhibitors are being examined in cellular studies, where certain of these agents have been shown to block hepatocyte growth factor (HGF)-induced cell migration in Met containing fibroblasts at nanomolar concentrations and to inhibit tubule formation potentially involved in angiogenesis. Using one of our agents, our collaborators have demonstrated inhibition of metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. This supports the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establishes a critical role for Grb2 SH2 domainmediated interactions in the metastatic process. More recently, we have undertaken a complimentary approach to blocking Grb2 function that does not target the SH2 domain. In this approach inhibitors are being developed that block the critical association of Grb2 with its constitutive binding partner, son-of-sevenless (SOS). This work involves the synthesis of peptides and peptide mimetics that bind to the Grb2 Src homology 3 (SH3) domain. Efforts have been been undertaken to develop SH2 domain-directed peptide mimetic inhibitors of Shc-dependent signaling. Shc proteins are non-catalytic SH2 domain-containing docking modules that participate in a variety of cell-regulatory processes associated with proliferation, survival and apoptosis. Shc as well as Grb2 proteins are particularly important for down stream signaling of PTKs, where they have been shown to link activation of the cytoplasmic kinase domains with Ras effectors. Shc has also been shown to serve as a critical angiogenic switch for for the production of vascular endothelial growth factor (VEGF) downstream from the c-Met and ErbB2 RTK oncoproteins, where recruitment of Shc but not Grb2 has been shown to be a required event. Accordingly, disruption of Shc-dependent signaling through blockade of its SH2 domain may afford a new therapeutic approach to cancers reliant on disregulation of such PTKs. It has previously been reported that the 14-mer zeta-chain-T cell receptor peptide, Ac-GHDGLpYQGLSTATK-amide (where pY = pTyr) binds to the Shc SH2 domain with an affinity of Kd = 50 μM. We prepared analogues that contained functionality not present in genetically-encoded amino acids, including N-alkylglycine (peptoid) residues. This work resulted in the discovery of tetrameric peptide peptoid hybrids that exhibit good Shc SH2 domain binding affinity. Further structural optimization of these agents is in progress. In order to conduct cell-based studies, membrane carrier peptide sequences were chemically linked to select high affinity phosphopeptides resulting from these efforts. Preliminary data has indicated that these peptides can block the binding of Shc to activated Met in whole cells at low micromolar concentrations. Overexpression of the serine/threonine polo-like kinase 1 (Plk1) is tightly associated with oncogenesis in several human cancers. Interference with Plk1 function induces apoptosis in tumor cells but not in normal cells. Accordingly, Plk1 is a potentially attractive anticancer chemotherapeutic target. Plk1 possesses a unique phosphopeptidebinding polo box domain (PBD) that is essential for its intracellular localization and mitotic functions. Unlike kinase domains, PBDs are found only in the four members of Plks. Therefore, they represent ideal targets for selectively inhibiting the function of Plks. By examining various PBD-binding phosphopeptides, our NCI collaborator, Dr. Kyung Lee, previously found that a 5mer phosphopeptide PLHSpT specifically interacts with the Plk1 PBD with high affinity, whereas it fails to significantly interact with the PBDs of two closely-related kinases, Plk2 and Plk3. Using a unique """"""""post solid-phase diversification"""""""" technique developed in our laboratory, we have optimized the binding affinity of a series of peptides and peptide mimetics. This has resulted in greater than 100-fold enhancement in PBD1 binding affinity. Work is in progress in the laboratory of Dr. Michael Yaffe (MIT) to solve the X-ray crystal structure of our high affinity ligands bound to PBD1 protein. The results of these studies should facilitate a further structure-based optimization of our PBD1-binding inhibitors. In the phosphatase area, inhibitors are being developed against the YopH PTP, which is a pathogenic component of the potential bioterrosim agent Yersinia pestis. This work is being done in collaboration with Drs. Robert Ulrich (USAMRIID) and David Waugh (NCI). A focused library approach has been used wherein two aromatic fragments are joined together by a series of linker segments. This has led to the identification of low micromolar affinity inhibitors that are undergoing further optimization. A parallel approach to inhibitor development is being conducted. The approach is unusual in that it relies on the optimization of YopH substrates to provide structural starting points for inhibitor development. Final inhibitors will be obtained by replacing the phosphate esters by hydrolytically stable bioisosteres. In the area of PTK inhibitor development, the high affinity Sugen Pharmaceuticals- c-Met kinase inhibitor SU11274 is being used as a starting point for structural elaboration. X-ray crystal studies have shown that c-Met binding interactions of SU11274 are confined well within the catalytic cleft. We have synthetically appended linker chains onto the SU11274 core to retain the original binding within the catalytic cleft while at the same time introduction additional binding interactions exterior to catalytic cleft. This work has the potential to advance the development of PTK inhibitors in general.
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