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：异常的 PK 依赖性信号传导与多种癌症的病因有关。因此，正在开发药物制剂来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成分来开发 PK 依赖性信号传导抑制剂：（1）通过 src 同源 2 (SH2) 结构域与磷酸酪氨酰 (pTyr) 残基的识别和结合介导的蛋白质-蛋白质关联； (2)通过细胞蛋白酪氨酸磷酸酶(PTP)去除pTyr磷酰基。 (3) Src 同源 3 (SH3) 结构域介导的蛋白质-蛋白质关联； (4) polo 样激酶 1 的 Polobox 结合结构域。 (1) SH2 结构域结合抑制剂。高亲和力生长因子受体结合蛋白 2 (Grb2) 结合拮抗剂正在准备作为 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 博士和 Marston Linehan 博士）合作的一部分，我们的 Grb2 信号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阻断肝细胞生长因子 (HGF) 诱导的细胞在纳摩尔浓度的含有 Met 的成纤维细胞中迁移，并抑制可能参与血管生成的小管形成。我们的合作者使用我们的一种药物，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 Grb2 SH2 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 是潜在生物病原体鼠疫耶尔森氏菌的致病成分。这项工作是与博士合作完成的。罗伯特·乌尔里希 (USAMRIID) 和戴维·沃 (David Waugh) (NCI)。已使用集中库方法，其中两个芳香族片段通过一系列接头片段连接在一起。这导致了低微摩尔亲和力抑制剂的鉴定，这些抑制剂正在进一步优化。正在开展抑制剂开发的并行方法，该方法依赖于 YopH 底物的优化，为抑制剂开发提供结构起点。该方法的独特之处在于使用硝基苯基磷酸酯底物，可以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。最终的抑制剂是通过用水解稳定的生物等排体代替磷酸酯获得的。这项工作产生了低纳摩尔非混杂抑制剂。 (3) SH3结构域结合抑制剂。我们已经开发了基于肽的抑制剂，可以阻断 Grb2 与其组成性结合伙伴 Son-of-Sevenless (SOS) 的关键关联。这项工作涉及与 Grb2 Src 同源 3 (SH3) 结构域结合的肽和肽模拟物的合成。闭环复分解 (RCM) 已用于制备大环肽，该肽表现出增强的阻断细胞裂解物中同源 Grb2-SOS 复合物形成的能力。 (4) Polo 样激酶 1 (Plk1) Polo Box 结构域结合抑制剂：丝氨酸/苏氨酸 Polo 样激酶 1 (Plk1) 的过度表达与多种人类癌症的肿瘤发生密切相关。干扰 Plk1 功能会诱导肿瘤细胞凋亡，但不会诱导正常细胞凋亡。因此，Plk1 是一个潜在有吸引力的抗癌化疗靶点。 Plk1 拥有独特的磷酸肽结合 polo box 结构域 (PBD)，这对于其细胞内定位和有丝分裂功能至关重要。与激酶结构域不同，PBD 仅存在于 Plk 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。通过检查各种 PBD 结合磷酸肽，我们的 NCI 合作者 Kyung Lee 博士之前发现，5 聚体磷酸肽 PLHSpT 与 Plk1 PBD 具有高亲和力特异性相互作用，而它无法与两个密切相关的激酶的 PBD 显着相互作用， Plk2 和 Plk3。从先前报道的含有 5-氨基酸 pThr 的肽开始，通过结构精炼的迭代顺序过程。与德国莱比青大学的 Thorsten Berg 博士合作确定的 Plk1、2 和 3 PBD 结构域的结合亲和力表明，我们已经能够将 Plk1 PBD 结合亲和力增加超过 3 个数量级，同时保留对 Plk1 PBD 具有高选择性，而不与相关的 Plk2 或 Plk3 PBD 结合。发现了三种不同类别的高亲和力结合抑制剂，其中含有新的且尚未报道的氨基酸类似物。与 Michael Yaffe 博士（麻省理工学院）合作，解析了这些与 Plk1 PBD 蛋白结合的肽的 X 射线共晶结构，结果显示揭示了一种以前从未观察到的完全意想不到的结合模式。这项工作开发了全新的氨基酸类似物，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，应重新定义 PBD 导向抑制剂领域。这些化合物有可能为新型抗癌化疗药物提供基础。