Inhibitors of Tyrosine Kinase-Dependent Signalling

正在开发药物来调节磷酸酪氨酸（pTyr）依赖的细胞信号传导。重点是通过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ptyr依赖性结合相互作用的抑制剂。在SH2结构域，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包括erbB-2和Met依赖性癌症在内的多种癌症的潜在新疗法进行。在此之前，我们已经制备了一系列对酶降解稳定的模拟ptyr的氨基酸类似物。它们被整合到三肽平台中，并显示出高Grb2-SH2结构域结合亲和力。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。在细胞外Grb2-SH2结构域结合实验中，这些新的大环化合物显示出显著增强的效力。对某些类似物，观察到结合常数在低皮摩尔范围内。这些类似物是目前报道的针对SH2结构域的最有效的合成拮抗剂。重要的是，在全细胞实验中，这些大环配体保持了优异的效力，对Grb2 SH2结构域结合的体内抑制常数在低纳摩尔范围内。在通过erB-2依赖途径利用Grb2驱动有丝分裂的乳腺癌细胞培养中，这些大环在亚微摩尔浓度下显示出细胞抑制作用。相同的化合物对不依赖Grb2途径存活的类似细胞无毒。在平行合作中，研究了依赖grb2依赖性信号通路的von Hippel-Lindau （VHL）依赖性肾癌的效应。在细胞研究中，这些药物在纳摩尔浓度下抑制肝细胞生长因子（HGF）诱导的含met成纤维细胞的细胞迁移，并抑制可能参与血管生成的小管形成。新的大环Grb2 SH2结构域信号抑制剂的设计、合成和生物学评价工作仍在继续。在磷酸酶领域，一种基于结构的PTP抑制剂设计方法正在进行中。以表皮生长因子受体（EGFr）自磷酸化位点衍生的三肽序列为展示平台，我们检测了一组合成的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。目前正在进行工作，以优化这些三肽引线作为治疗鼠疫的潜在疗法。这种治疗方法可能对使用鼠疫耶尔森菌作为生物恐怖制剂有价值。

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. Previously we had prepared a series of pTyr-mimicking amino acid analogues that were stable toward enzymatic degradation. These were incorporated into tripeptide platforms and shown to have high Grb2-SH2 domain-binding affinity. During the reporting period novel macrocycles were prepared that represented conformationally constrained tetrapeptide-mimicking variants of our earlier tripeptide inhibitors. In extracellular Grb2-SH2 domain binding assays, these new macrocyclic compounds displayed significantly enhanced potency. For certain analogues, binding constants in the low picomolar range were observed. These analogues represent the most potent synthetic antagonists reported against any SH2 domain. Importantly, in whole cell assays these macrocyclic ligands maintained superior potency, with in vivo inhibition constants against Grb2 SH2 domain binding being in the low nanomolar range. In culture against breast cancer cells that are mitogenically-driven through erB-2 dependent pathways that utilize Grb2, these macrocycles displayed cytostatic effects at submicromolar concentrations. The same compounds were non-toxic against similar cells that are not dependent on Grb2 pathways for survival. In a parallel collaboration effects were studied against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb2-dependent signaling pathways. In cellular studies, 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. Work continues on the design, synthesis and biological evaluation of new macrocyclic Grb2 SH2 domain-signaling inhibitors. In the phosphatase area, a structure-based approach toward PTP inhibitor design is being pursued. Using as a display platform, a tripeptide sequence derived from an epidermal growth factor receptor (EGFr) autophosphorylation site, we 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 is ongoing to optimize these tripeptide leads as potential therapeutics for the treatment of plague. Such therapeutics may have value against the use of Yersinia pestis as a bioterrorism agent.