Inhibitors of Tyrosine Kinase-Dependent Signalling as An

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。这两项努力的核心是开发新的pTyr模拟物，这种模拟物要么对PTPs的酶降解提供更高的稳定性，要么提供更高的亲和力。在SH2结构域，细胞可穿透生长因子受体结合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。我们在这方面的努力一直在继续，制备了一种新的pTyr模拟物，用稳定的亚甲基取代了不稳定的磷酸酯键，用羧亚甲基取代了α-氮官能团。当与合适的三肽平台结合时，这种新的氨基酸类似物在细胞外和全细胞检测中都显示出优越的效力。在本报告所述期间，我们制备了新的大环肽模拟物，它们代表了我们早期三肽抑制剂的构象受限变体。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，在接近皮摩尔范围内观察到结合常数。重要的是，在全细胞分析中，这些大环配体保持了增强的效力。体内对Grb2SH2结构域结合的抑制常数在亚微摩尔范围内。在对抗乳腺癌细胞的培养中，这些大周期显示出良好的细胞抑制作用，这些细胞通过Erb-2依赖的途径利用Grb2进行有丝分裂驱动。相同的化合物对不依赖Grb2途径生存的相似细胞无毒。目前正在进行合作研究，以检查这些药物针对乳腺癌的联合治疗。平行合作正在研究对抗von Hippel-Lindau(VHL)依赖的肾癌的效应剂，这种肾癌依赖于Grb2依赖的信号通路。我们的大环化策略的成功促使我们探索替代的大环化技术。这包括新的氮杂多肽大环，其中关键的碳原子被氮取代。在另一种方法中，第一个已报道的β-氨基pTyr模拟物被制备出来，目前正在用于制备一类新的β-氨基大环化类似物。在磷酸酶领域，正在寻求基于结构的抑制剂设计方法。利用表皮生长因子受体(EGFR)衍生的含有pTyr的多肽序列作为平台，我们先前已经描述了一些新的非含磷的pTyr模拟物对PTP1B的抑制效力。以这种方式确定的高度有效的基序已经成为小分子多肽模拟设计的模型。在报告所述期间，开始了开发YopH抑制剂的新努力，YopH是鼠疫耶尔森菌的一种PTP活性致病成分。这种努力的前提是需要保护剂，防止可能将鼠疫耶尔森菌用作生物恐怖主义有机体。到目前为止，使用新的pTyr模拟物，已经鉴定出对YopH表现出低微摩尔亲和力的三肽抑制剂。有重点的图书馆正在进行优化这些线索。作为这项工作的一部分，正在进行一项合作，以确定我们的抑制剂与YopH酶共结晶的X射线晶体结构。从这些研究获得的信息将被用于基于结构的设计，将模拟肽的YopH抑制剂作为潜在的YopH暴露保护剂。

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. Central to both of these efforts is the development of new pTyr mimetics which afford either increased stability toward enzymatic degradation by PTPs or increased affinity. In the SH2 domain area, development of cell-permeable growth factor receptor-bound 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. Our efforts in this area have continued with the preparation of a new pTyr mimetic having both the labile phosphate ester bond replaced with a stable methylene group and the alpha-nitrogen functionality replaced with a carboxymethylene moiety. When incorporated into an appropriate tripeptide platform, this novel amino acid analogue exhibits superior potency in both extracellular and whole cell assays. During the reporting period novel macrocyclic peptide mimetics were prepared that represent conformationally constrained 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 near picomolar range were observed. Importantly, in whole cell assays these macrocyclic ligands maintained enhanced potency. In vivo inhibition constants against Grb2 SH2 domain binding were in the sub-micromolar range. In culture against breast cancer cells mitogenically driven through erB-2 dependent pathways that utilize Grb2, these macrocycles displayed good cytostatic effects. The same compounds were non-toxic against similar cells that are not dependent on Grb2 pathways for survival. Collaborative studies are currently underway to examine these agents in combination therapies directed against breast cancer. Parallel collaborations are studying effects agents against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb2-dependent signaling pathways. The success of our macrocyclization strategy has lead us to explore alternate macrocyclization techniques. This has included novel aza-peptide macrocycles in which critical carbon atoms are replaced by nitrogens. In another approach, the first beta-amino pTyr mimetic yet reported was prepared and is currently being utilized to prepare a new family of beta-amido macrocyclized analogues.In the phosphatase area, a structure-based approach toward inhibitor design is being pursued. Using an epidermal growth factor receptor (EGFr)-derived pTyr-containing peptide sequence as a platform, we had previously delineated a number of novel non phosphorus containing pTyr mimetics for inhibitory potency against PTP1B. Highly potent motifs identified in this fashion have served as models for small molecule peptidomimetic design. During the reporting period a new effort was begun to develop inhibitors of YopH, a PTP active pathogenic component of Yersinia pestis. Such efforts are predicated on the need for protective agents against potential use of Yersinia pestis as a bioterrorism organism. To date using novel pTyr mimetics, tripeptide inhibitors have been identified that exhibit low micromolar affinity against YopH. Focussed libraries are in progress to optimize these leads. As part of this effort, a collaboration is in place determine X-ray crystal structures ofour inhibitors co-crystallized with the YopH enzyme. Information derived from these studies will be used for the structure-based design of peptidomimetic YopH inhibitors as potential protective agents against YopH exposure.