Pharmacological agents are being developed to modulate phosphotyrosyl (pTyr) dependent cell signalling. Particular emphasis is on interfacial inhibitors of pTyr dependent binding interactions, which are mediated by src homology 2 (SH2) domains. Secondary efforts are being directed at protein tyrosine phosphatase (PTP) inhibitors.In the SH2 domain area, high affinity growth factor receptor-bound protein 2 (Grb2)-binding antagonists are being developed as potential new therapeutics for a variety of cancers including 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 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 ongoing. We have prepared biotinylated variants of certain of our Grb2 SH2 domain signaling inhibitors that are being used as pharmacological tools to identify intracellular targets. These agents include the highest affinity biotinylated SH2 domain-binding antagonist yet reported. We had previously reported using ring-closing metathesis (RCM) to prepare novel macrocycles designed as conformationally constrained peptide mimetics of our open-chain tripeptide inhibitors. The high synthetic complexity of these macrocyclic peptide mimetics was in part due to the stereoselective construction of a key upper ring-forming junction. In order to simplify synthesis, new macrocycle variants were prepared bearing upper ring junctions that (1) were prepared non-stereoselectively; (2) utilized commercially-available allylglycine residue or (3) employed symmetrical achiral moieties. In all three approaches, high Grb2 SH2 domain-binding affinities were obtained. Of particular note for approach (3) was near total retention of binding affinity of the parent stereoselectively-formed macrocycle. In an effort to explore and extend our RCM macrocyclization approach we examined [2+3] azide - alkyne cycloaddition """"""""click chemistry"""""""" reactions and ring closure using beta-aminomethylene groups. In the case of click chemistry ring closure, high affinity binding antagonists were obtained. These investigations have advanced the field of macrocyclic peptidomimetic synthesis. Efforts were also 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 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. It has previously been reported that the 14-mer zeta-chain-T cell receptor pY141 peptide, """"""""Ac-GHDGLpYQGLSTATK-amide"""""""" binds to the Shc SH2 domain with an affinity of Kd = 50 iM. Using the published crystal structure of the Shc SH2 domain, molecular modeling studies have indicated that significant interactions only occur for the sequence """"""""pYQGL."""""""" Accordingly, we prepared the hexamer partial sequence """"""""Ac-LpYQGLS-amide"""""""" and found it to exhibit KD = 84 iM in a Biacore binding assay.
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