Inhibitors of Tyrosine Kinase-Dependent Signalling as An

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Pharmacological agents are being developed to modulate phosphotyrosyl (pTyr) dependent cell signalling. Emphasis is on inhibitors of pTyr dependent binding interactions, which are mediated by src homology 2 (SH2) domains and on protein tyrosine phosphatase (PTP) inhibitors. In the SH2 domain area, development of cell permeable growth factor receptor-bound protein 2 (Grb2) antagonists is being undertaken as potential new therapeutics for a variety of cancers including erbB-2 and Met dependent cancers. During the reporting period novel macrocycles were prepared that represented conformationally constrained tetrapeptide-mimicking variants of our earlier tripeptide inhibitors. In an effort to explore and extend the macrocyclization approach a variety of new chemistries were examined. These include a several different olefin methasis reactions, [2+3] azide - alkyne cycloaddition reactions and ring closure using beta-aminomethylene groups. These investigations have advanced the field of macrocyclic peptidomimetic synthesis. As part of a collaborative effort with NCI clinical investigators, Grb2 signaling inhibitors were studied against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb dependent signaling pathways. In cellular studies, certain of these agents inhibit hepatocyte growth factor (HGF)-induced cell migration in Met containing fibroblasts at nanomolar concentrations and inhibit tubule formation potentially involved in angiogenesis. Metastasis model animal studies are currently ongoing. Biotinylated variants of select potent Grb2 SH2 domain signaling inhibitors were also prepared and are being used as pharmacological tools to identify intracellular targets. Efforts were also begun 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 receptor tyrosine kinases (RTKs), 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 VEGF production downstream from the 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 interactions may afford a new therapeutic approach to cancers reliant on disregulation of such RTKs. In the phosphatase area, a structure-based approach toward PTP inhibitor design has been pursued. Using as a display platform, a tripeptide sequence derived from an epidermal growth factor receptor (EGFr) autophosphorylation site, we had previously examined a panel of synthetic pTyr mimetics for inhibitory potencies against YopH, which is a pathogenic PTP component of Yersinia pestis, the causative agent of plague. Certain of these tripeptides exhibited binding constants in the single-digit micromolar range. Work during the reporting period continued to optimize these tripeptide leads as potential therapeutics for the treatment of plague. Currently, known high affinity YopH inhibitors are being prepared for co-crystallography with the YopH protein and X-ray crystallographic structure determination. Information gained in this way will be used to design focused libraries of inhibitors. Inhibitors derived from this work may have therapeutic value against the use of Yersinia pestis as a bioterrorism agent.