Inhibitors of Tyrosine Kinase-Dependent Signalling

正在开发药物来调节依赖于磷酸酪氨酰(PTyr)的细胞信号转导。重点是pTyr依赖的结合作用的抑制剂，这是由src同源2(SH2)结构域和蛋白质酪氨酸磷酸酶(PTP)抑制剂介导的。在SH2结构域，细胞可穿透生长因子受体结合蛋白2(Grb2)拮抗剂的开发被认为是治疗多种癌症的潜在新疗法，包括erbB-2和Met依赖型癌症。在此之前，我们已经制备了一系列对酶降解稳定的pTyr类似物。这些化合物被整合到三肽平台上，并显示出高的Grb2-SH2结构域结合亲和力。在报告期间，制备了新的大环，它们代表了我们早期三肽抑制剂的构象受限的四肽仿制变体。在细胞外Grb2-SH2结构域结合分析中，这些新的大环化合物显示出显著增强的效力。对于某些类似物，在较低的皮摩尔范围内观察到结合常数。这些类似物代表了已报道的针对SH2结构域的最有效的合成拮抗剂。重要的是，在全细胞分析中，这些大环配体保持了优越的效力，体内对Grb2 SH2结构域结合的抑制常数处于低纳摩尔范围。在针对乳腺癌细胞的培养中，这些大周期在亚微摩尔浓度下显示出细胞抑制作用，这些细胞是通过利用Grb2的Erb-2依赖途径进行有丝分裂驱动的。相同的化合物对不依赖Grb2途径生存的相似细胞无毒。在一项平行的合作中，研究了对von Hippel-Lindau(VHL)依赖的肾癌的影响，这些肾癌依赖于Grb2依赖的信号通路。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Grb2SH2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，正在寻求一种基于结构的方法来设计PTP抑制剂。以来自表皮生长因子受体(EGFR)自身磷酸化位点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的抑制效力，YopH是鼠疫耶尔森氏菌的致病PTP成分，鼠疫病原体。其中某些三肽的结合常数在一位数微摩尔范围内。目前正在进行优化这些三肽先导化合物作为治疗鼠疫的潜在疗法的工作。这种疗法可能对将鼠疫耶尔森氏菌用作生物恐怖主义制剂具有价值。

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.