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

酪氨酸激酶依赖性信号传导抑制剂作为抗癌药物

• 批准号: 8348901
• 负责人: TERRENCE BURKE
• 金额: $ 96.5万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8348901
• 关键词: Affect; Affinity; Amino Acid Sequence; Amino Acids; Antineoplastic Agents; Apoptosis; Binding; Biological Availability; Cells; Clinical Investigator; Collaborations; Color; Complex; Cytotoxic agent; Development; Docking; ERBB2 gene; Etiology; Excision; Exhibits; Fibroblasts; Germany; Growth Factor Receptors; Hepatocyte Growth Factor; Human; Immunoglobulin Fragments; Libraries; Malignant Neoplasms; Measurement; Mediating; Mitotic; Modeling; Monitor; Mus; Neoplasm Metastasis; Nitrophenol; Normal Cell; Peptide Sequence Determination; Peptide Synthesis; Peptides; Pharmaceutical Preparations; Phosphopeptides; Phosphotransferases; Plague; Polo-Box Domain; Primary Neoplasm; Process; Protein Kinase; Protein Tyrosine Phosphatase; Proteins; Reaction; Reporting; Roentgen Rays; Role; Series; Serine; Signal Transduction; Solid Neoplasm; Son; Therapeutic; Threonine; Tyrosine Kinase Inhibitor; Universities; Work; Yersinia pestis; Yersinia yopH protein; analog; angiogenesis; anti-cancer therapeutic; base; cell motility; cytotoxic; design; growth factor receptor-bound protein 2; human PLK1 protein; in vivo; inhibitor/antagonist; meetings; member; metastatic process; mimetics; neoplastic cell; nitrophenylphosphate; novel; novel therapeutics; overexpression; peptide structure; phosphatase inhibitor; phosphate ester; receptor binding; small molecule; tumor; tumor growth; tumorigenesis

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.

靶向蛋白激酶（PK）依赖性信号传导：异常PK-抑素信号传导与几种癌症的病因相关。因此，正在开发药理学剂来调节激酶依赖性信号作为潜在的新抗癌疗法。我们通过靶向四个关键成分来开发依赖PK的信号传导抑制剂：（1）通过识别和结合SRC同源性2（SH2）结构域与磷酸酪糖基（PTYR）残基介导的蛋白质 - 蛋白质缔合； （2）通过细胞蛋白 - 酪氨酸磷酸酶（PTPS）去除PTYR磷酸基团。 （3）SRC同源性3（SH3）结构域介导的蛋白质蛋白相关； （4）polo样激酶的polobox结合结构域1。（1）SH2结构域结合抑制剂。高亲和力生长因子受体结合的蛋白2（GRB2）结合拮抗剂是作为ERBB-2和C-MET依赖性癌症的潜在新疗法准备的。作为与NCI临床研究人员（Don Bottaro博士和Marston Linehan）的合作努力的一部分，在细胞研究中正在检查我们的GRB2信号抑制剂，在细胞研究中，这些药物中的某些药物被证明可以阻止肝细胞生长因子（HGF）在含有纤维分类的拟合纤维群中的细胞迁移，并在Nananomomomomomololts的含量中迁移，并在Nanomomolol oltim promentation inanom oltim promistitions中添加了对杂质的迁移。使用我们的一种药物，我们的合作者在两个侵袭性肿瘤模型中表现出了体内转移的抑制，而不会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的转移扩散方面的潜在疗效，并确立了GRB2 SH2 SH2域介导的相互作用在转移过程中的关键作用。 （2）PTP抑制剂：正在针对Yoph PTP开发合成的小分子抑制剂，这是潜在的生物素剂耶尔森氏菌的致病成分。这项工作是与Drs合作完成的。罗伯特·乌尔里希（Robert Ulrich）（Usamriid）和大卫·沃（David Waugh）（NCI）。已经使用了一种集中的库方法，其中两个芳香片段通过一系列的链接段将其连接在一起。这导致了正在进行进一步优化的低微摩尔亲和力抑制剂的鉴定。正在进行一种平行的抑制剂开发方法，依赖于YOPH底物的优化为抑制剂开发提供结构起点。该方法在使用硝基苯基磷酸底物方面是独一无二的，该基材允许通过简单测量黄色衍生反应产物硝基苯酚来监测底物释放。最终抑制剂是通过用水解稳定的生物酶代替磷酸酯来获得的。这项工作产生了低纳米摩尔的非乳腺抑制剂。 （3）SH3结构域结合抑制剂。我们已经开发了基于肽的抑制剂的发展，这些抑制剂阻止了GRB2与其构成结合伴侣的七个儿子（SOS）的关键关联。这项工作涉及与GRB2 SRC同源性3（SH3）结构域结合的肽和肽模拟物的合成。插环的分解（RCM）已用于制备大环肽，该肽表现出增强的能力阻断细胞裂解物中同源GRB2-SOS复合物的形成。 （4）polo样激酶1（PLK1）polo Box结构域结合抑制剂：丝氨酸/苏氨酸类球样激酶1（PLK1）的过表达与几种人类癌症中的肿瘤发生密切相关。对PLK1功能的干扰会诱导肿瘤细胞的凋亡，但在正常细胞中不引起凋亡。因此，PLK1是一个潜在吸引人的抗癌化学治疗靶标。 PLK1具有独特的磷酸肽固定polo盒结构域（PBD），这对于其细胞内定位和有丝分裂功能至关重要。与激酶域不同，PBD仅在PLK的四个成员中发现。因此，它们代表了选择性抑制PLK功能的理想目标。通过检查各种PBD结合磷酸肽，我们的NCI合作者Kyung Lee博士先前发现，5mer磷酸肽PLHSPT与具有高亲和力的PLK1 PBD特别相互作用，而它与两个紧密相关的Kinass assepass，PLK2和PLK2和PLK3和PLK3和PLK3和PLK3和PLK3的PBD相互作用。从先前报道的5-含有肽的5-氨基酸PTHR开始，从结构改进的迭代顺序过程开始。与PLK1、2和3的PBD域的结合亲和力与德国莱比锡大学的Thorsten Berg博士确定，这表明我们能够将PLK1 PBD结合亲和力提高3个范围以上，同时保持PLK1 PBD的高度选择性，而无需与相关的PLK1 PBD保持高选择性。发现了三种不同的高亲和力结合抑制剂，其中包含新的且尚未报告的氨基酸类似物。与迈克尔·亚菲（Michael Yaffe）博士（MIT）X射线X射线共结构结构合作，已证明与PLK1 PBD蛋白结合的这些肽结构显示，显示出一种完全意外的结合模式，这种结合模式以前从未观察到过。这项工作导致了全新的氨基酸类似物的发展，并将其应用于三类选择性，高亲和力PLK1 PBD抑制剂。这些抑制剂表现出的独特结合模式定义了一种全新的PBD结合相互作用类型，该类型应该重新定义PBD定向抑制剂的领域。这些化合物有可能为新型抗癌化学治疗的基础提供基础。