Inhibitors of Tyrosine Kinase-Dependent Signalling as An

正在开发药物来调节磷酸酪氨酸（pTyr）依赖的细胞信号传导。重点是pTyr依赖性结合相互作用的抑制剂，这是由src同源性2 （SH2）结构域和蛋白酪氨酸磷酸酶（PTP）抑制剂介导的。在SH2区域，细胞渗透性生长因子受体结合蛋白2 （Grb2）拮抗剂的开发正在作为多种癌症的潜在新疗法进行，包括erbB-2和Met依赖性癌症。在报告期间，制备了新的大环，代表了我们早期三肽抑制剂的构象约束的四肽模拟变体。为了探索和扩展大环化方法，研究了各种新的化学物质。这些反应包括几种不同的烯烃甲基化反应，[2+3]叠氮化物-炔环加成反应和使用-氨基甲基闭合环。这些研究促进了大环拟肽合成领域的发展。作为与NCI临床研究人员合作的一部分，研究了Grb2信号抑制剂对依赖Grb信号通路的von Hippel-Lindau （VHL）依赖性肾癌的治疗作用。在细胞研究中，某些药物抑制肝细胞生长因子（HGF）诱导的细胞在含纳米摩尔浓度的Met中的迁移，并抑制可能参与血管生成的小管形成。转移模型动物研究目前正在进行中。生物素化变异的选择性强效Grb2 SH2域信号抑制剂也被制备出来，并被用作鉴定细胞内靶点的药理学工具。人们也开始努力开发SH2结构域导向的shc依赖性信号的肽模拟抑制剂。Shc蛋白是一种非催化SH2结构域对接模块，参与多种与增殖、存活和凋亡相关的细胞调控过程。Shc和Grb2蛋白对于受体酪氨酸激酶（rtk）的下游信号传导尤其重要，它们已被证明将细胞质激酶结构域的激活与Ras效应物联系起来。Shc也被证明是Met和ErbB2 RTK癌蛋白下游VEGF生成的关键血管生成开关，其中Shc而不是Grb2的募集已被证明是必需的事件。因此，通过阻断其SH2结构域相互作用来破坏shc依赖性信号可能为依赖于这些rtk失调的癌症提供新的治疗方法。在磷酸酶领域，一种基于结构的PTP抑制剂设计方法一直在进行。我们利用表皮生长因子受体（EGFr）自磷酸化位点衍生的三肽序列作为展示平台，检测了一组合成的pTyr模拟物对YopH的抑制能力，YopH是鼠疫病原体鼠疫耶尔森菌的一种致病性PTP成分。其中某些三肽的结合常数在个位数微摩尔范围内。本报告所述期间的工作继续优化这些三肽引线作为治疗鼠疫的潜在疗法。目前，已知的高亲和力YopH抑制剂正在制备用于与YopH蛋白共晶学和x射线晶体学结构测定。以这种方式获得的信息将用于设计抑制剂的重点文库。从这项工作中获得的抑制剂可能具有治疗鼠疫耶尔森氏菌作为生物恐怖剂的价值。

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.