Inhibitors of Tyrosine Kinase-Dependent Signalling as Anti-Cancer Agents

靶向蛋白激酶（PK）依赖性信号传导：异常的PK依赖性信号传导与几种癌症的病因学相关。为此，正在开发药理学试剂以调节激酶依赖性信号传导作为潜在的新抗癌疗法。我们正在通过靶向四个关键组分开发PK依赖性信号传导抑制剂：（1）通过src同源2（SH 2）结构域与磷酸酪氨酸（pTyr）残基的识别和结合介导的蛋白质-蛋白质缔合;（2）通过细胞蛋白酪氨酸磷酸酶（PTP）去除pTyr磷酸基团。(3)Src同源性3（SH 3）结构域介导的蛋白质-蛋白质缔合;（4）polo样激酶1的Polobox结合结构域。(1)SH 2结构域结合抑制剂。高亲和力生长因子受体结合蛋白2（Grb 2）结合拮抗剂正被制备作为erbB-2和c-Met依赖性癌症的潜在新疗法。作为与NCI临床研究人员（Don Bottaro和马斯顿Linehan博士）合作的一部分，我们的Grb 2信号传导抑制剂正在细胞研究中进行检查，其中某些药物已被证明可以在纳摩尔浓度下阻断含Met成纤维细胞中肝细胞生长因子（HGF）诱导的细胞迁移，并抑制可能参与血管生成的小管形成。使用我们的一种药物，我们的合作者已经在两种侵袭性肿瘤模型中证明了体内转移的抑制作用，而不影响原发性肿瘤的生长速率。这支持了该化合物在减少原发性实体瘤转移扩散方面的潜在功效，并确立了Grb 2 SH 2结构域介导的相互作用在转移过程中的关键作用。(2)PTP抑制剂：正在开发针对YopH PTP的合成小分子抑制剂，YopH PTP是潜在的生物恐怖剂鼠疫耶尔森氏菌的致病成分。这项工作是与Robert Ulrich博士（USAMRIID）和大卫沃博士（NCI）合作完成的。已经使用了聚焦文库方法，其中两个芳族片段通过一系列接头区段连接在一起。这导致了低微摩尔亲和力抑制剂的鉴定，正在进行进一步优化。正在进行抑制剂开发的平行方法，其依赖于优化YopH底物以提供抑制剂开发的结构起点。该方法的独特之处在于其使用硝基苯磷酸盐底物，其允许通过简单测量黄色衍生的反应产物硝基酚来监测底物释放。最终抑制剂通过用水解稳定的生物电子等排体代替磷酸酯而获得。这项工作已经产生了低纳摩尔的非混杂抑制剂。 (3)SH 3结构域结合抑制剂。我们已经着手开发基于肽的抑制剂，其阻断Grb 2与其组成性结合伴侣Son-of-Sevenless（SOS）的关键关联。这项工作涉及的肽和肽模拟物，结合Grb 2 Src同源3（SH 3）结构域的合成。闭环复分解（RCM）已被用于制备大环肽，其表现出增强的阻断细胞裂解物中同源Grb 2-SOS复合物形成的能力。(4)Polo样激酶1（Plk 1）波罗盒结构域结合抑制剂：丝氨酸/苏氨酸Polo样激酶1（Plk 1）的过表达与几种人类癌症的肿瘤发生密切相关。干扰Plk 1功能可诱导肿瘤细胞凋亡，但不诱导正常细胞凋亡。因此，Plk 1是一个潜在的有吸引力的抗癌化疗靶点。Plk 1具有独特的磷酸肽结合波罗box结构域（PBD），其对于其细胞内定位和有丝分裂功能是必需的。与激酶结构域不同，PBD仅存在于Plks的四个成员中。因此，它们代表了选择性抑制Plks功能的理想靶标。通过研究各种PBD结合磷酸肽，我们的NCI合作者Kyung Lee博士先前发现，5聚体磷酸肽PLHSpT以高亲和力与Plk 1 PBD特异性相互作用，而它不能与两种密切相关的激酶Plk 2和Plk 3的PBD显着相互作用。从先前报道的含有5 -氨基酸pThr的肽开始，通过结构优化的迭代顺序过程。与Thorsten贝格博士，The University of Leipzing，德国合作测定的对Plk 1、2和3的PBD结构域的结合亲和力表明，我们已经能够将Plk 1 PBD结合亲和力增加超过3个数量级，同时保持对Plk 1 PBD的高选择性，而不结合相关的Plk 2或Plk 3 PBD。发现了三种不同类型的高亲和力结合抑制剂，其中含有新的和尚未报道的氨基酸类似物。与Michael Yaffe博士（麻省理工学院）合作，解决了与Plk 1 PBD蛋白结合的这些肽的X射线共晶体结构，揭示了一种以前从未观察到的完全出乎意料的结合模式。这项工作导致了全新的氨基酸类似物的开发及其在三类选择性高亲和力Plk 1 PBD抑制剂中的应用。这些抑制剂所表现出的独特的结合模式定义了一个全新的PBD结合相互作用的类型，应该重新定义PBD定向抑制剂的领域。这些化合物有可能成为新型的抗癌药物。

Targeting Protein- Kinase (PK)-Dependent Signaling: Aberrant PK-depenent signaling is associated with the etiology of several cancers. For this reason, pharmacological agents are being developed to modulate kinase-dependent signaling as potential new anticancer therapeutics. We are developing PK-dependent signaling inhibitors by targeting four critical components: (1) Protein-protein associations mediated by recognition and binding of src homology 2 (SH2) domains to phosphotyrosyl (pTyr) residues; (2) The removal of the pTyr phosphoryl group by cellular protein-tyrosine phosphatases (PTPs). (3) Src homology 3 (SH3) domain-mediated protein-protein associations; (4) Polobox binding domain of polo-like kinase 1. (1) SH2 Domain-Binding Inhibitors. High affinity growth factor receptor-bound protein 2 (Grb2)-binding antagonists are being prepared as potential new therapeutics for erbB-2 and c-Met dependent cancers. As part of a collaborative effort with NCI clinical investigators (Drs. Don Bottaro and Marston Linehan), our Grb2 signaling inhibitors are being examined in cellular studies, where certain of these agents have been shown to block hepatocyte growth factor (HGF)-induced cell migration in Met containing fibroblasts at nanomolar concentrations and to inhibit tubule formation potentially involved in angiogenesis. Using one of our agents, our collaborators have demonstrated inhibition of metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. This supports the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establishes a critical role for Grb2 SH2 domainmediated interactions in the metastatic process. (2) PTP Inhibitors: Synthetic small molecule inhibitors are being developed against the YopH PTP, which is a pathogenic component of the potential bioterrosim agent Yersinia pestis. This work is being done in collaboration with Drs. Robert Ulrich (USAMRIID) and David Waugh (NCI). A focused library approach has been used wherein two aromatic fragments are joined together by a series of linker segments. This has led to the identification of low micromolar affinity inhibitors that are undergoing further optimization. A parallel approach to inhibitor development is being conducted that relies on the optimization of YopH substrates to provide structural starting points for inhibitor development. The approach is unique in its use of nitrophenylphosphate substrates that allow the monitoring of substrate release by the simple measurement of yellow color derived reaction product nitrophenols. Final inhibitors are obtained by replacing the phosphate esters with hydrolytically-stable bioisosteres. This work has yielded low-nanomolar non-promiscuous inhibitors. (3) SH3 Domain-Binding Inhibitors. We have undertaken the development of peptide-based inhibitors that block the critical association of Grb2 with its constitutive binding partner, Son-of-Sevenless (SOS). This work involves the synthesis of peptides and peptide mimetics that bind to the Grb2 Src homology 3 (SH3) domain. Ring-closing metathesis (RCM) has been used to prepare macrocyclic peptide that exhibit enhanced ability to block the formation of cognate Grb2-SOS complexes in cell lysates. (4) Polo-like Kinase 1 (Plk1) Polo Box Domain Binding Inhibitors: Overexpression of the serine/threonine polo-like kinase 1 (Plk1) is tightly associated with oncogenesis in several human cancers. Interference with Plk1 function induces apoptosis in tumor cells but not in normal cells. Accordingly, Plk1 is a potentially attractive anticancer chemotherapeutic target. Plk1 possesses a unique phosphopeptidebinding polo box domain (PBD) that is essential for its intracellular localization and mitotic functions. Unlike kinase domains, PBDs are found only in the four members of Plks. Therefore, they represent ideal targets for selectively inhibiting the function of Plks. By examining various PBD-binding phosphopeptides, our NCI collaborator, Dr. Kyung Lee, previously found that a 5mer phosphopeptide PLHSpT specifically interacts with the Plk1 PBD with high affinity, whereas it fails to significantly interact with the PBDs of two closely-related kinases, Plk2 and Plk3. Starting from a previously reported 5 - amino acid pThr containing peptide, through an iterative sequential process of structural refinement. Binding affinities against the PBD domains of Plk1, 2 and 3 determined in collaboration with Dr. Thorsten Berg, The University of Leipzing, Germany, show that we have been able to increase the Plk1 PBD binding affinity by over 3 - orders of magnitude while retaining high selectivity for the Plk1 PBD without binding to the related Plk2 or Plk3 PBDs. Three distinct classes of high affinity-binding inhibitors were discovered, which contain new and as yet unreported amino acid analogues. In collaboration with Dr. Michael Yaffe (MIT) X-ray co-crystal structures of these peptides bound to Plk1 PBD protein were solved shown to reveal an entirely unanticipated mode of binding that has never been observed before. The work has resulted in the development of entirely new amino acid analogues and their application to three classes of selective, high affinity Plk1 PBD inhibitors. Unique binding modes exhibited by these inhibitors define an entirely new genre of PBD-binding interactions that should redefine the field of PBD-directed inhibitors. These compounds could potentially provide the basis for a new type anticancer chemotherapeutic.