Inhibitors of Tyrosine Kinase-Dependent Signalling as An

正在开发用于调节磷酸酪氨酸（pTyr）依赖性细胞信号传导的药理学试剂。重点是由src同源性2（SH 2）结构域介导的pTyr依赖性结合相互作用的抑制剂和蛋白酪氨酸磷酸酶（PTP）抑制剂。这两种努力的核心是开发新的pTyr模拟物，其提供对PTP的酶促降解的增加的稳定性或增加的亲和力。在SH 2结构域领域，正在开发细胞渗透性生长因子受体结合2（Grb 2）拮抗剂，作为多种癌症（包括erbB-2和Met依赖性癌症）的潜在新疗法。之前，我们已经制备了一系列对酶促降解稳定的pTyr模拟氨基酸类似物。这些被掺入三肽平台，并显示具有高Grb 2-SH 2结构域结合亲和力。我们在这一领域的努力一直在继续，制备了一种新的pTyr模拟物，其不稳定的磷酸酯键被稳定的亚甲基取代，α-氮官能团被羧基亚甲基部分取代。当掺入适当的三肽平台时，这种新型氨基酸类似物在细胞外和全细胞测定中均表现出上级效力。在报告期间，制备了代表我们早期三肽抑制剂的构象受限变体的新型大环肽模拟物。在细胞外Grb 2-SH 2结构域结合试验中，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，观察到在近皮摩尔范围内的结合常数。重要的是，在全细胞测定中，这些大环配体保持增强的效力。对Grb 2 SH 2结构域结合的体内抑制常数在亚微摩尔范围内。在针对乳腺癌细胞的培养中，这些大环化合物显示出良好的细胞生长抑制作用，所述乳腺癌细胞通过利用Grb 2的erB-2依赖性途径促有丝分裂驱动。相同的化合物对不依赖Grb 2途径生存的类似细胞无毒。目前正在进行合作研究，以检查这些药物在针对乳腺癌的联合治疗中的作用。平行合作正在研究针对依赖Grb 2依赖性信号通路的von Hippel-Lindau（VHL）依赖性肾癌的效应剂。我们的大环化策略的成功使我们探索替代的大环化技术。这包括新的氮杂肽大环化合物，其中关键的碳原子被氮取代。在另一种方法中，制备了第一个β-氨基pTyr模拟物，目前正用于制备一个新的β-氨基大环化类似物家族。使用表皮生长因子受体（EGFr）衍生的含pTyr的肽序列作为平台，我们先前已经描绘了许多新的不含磷的pTyr模拟物，用于对PTP 1B的抑制效力。以这种方式鉴定的高度有效的基序已经用作小分子肽模拟物设计的模型。在本报告所述期间，开始了一项新的努力，以开发YopH的抑制剂，YopH是鼠疫耶尔森氏菌的PTP活性致病成分。这些努力是基于对预防鼠疫耶尔森氏菌作为生物恐怖主义生物体的潜在用途的保护剂的需要。迄今为止，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。重点图书馆正在进行中，以优化这些线索。作为这项工作的一部分，一项合作正在进行，以确定与YopH酶共结晶的抑制剂的X射线晶体结构。从这些研究中获得的信息将用于基于结构的拟肽YopH抑制剂的设计，作为潜在的保护剂对YopH暴露。

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.