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依赖性信号传导与几种癌症的病因有关。由于这个原因，正在开发药物来调节激酶依赖性信号作为潜在的新的抗癌治疗方法。我们正在通过针对四个关键成分开发pk依赖性信号抑制剂：(1)src同源性2 （SH2）结构域与磷酸酪氨酸基（pTyr）残基的识别和结合介导的蛋白-蛋白关联；(2)细胞蛋白酪氨酸磷酸酶（ptp）去除pTyr磷酸化基。(3) Src同源3 （SH3）结构域介导的蛋白关联；(4) polo样激酶1的Polobox结合域。(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）结合拮抗剂正被制备为erbB-2和c-Met依赖性癌症的潜在新疗法。作为与NCI临床研究者(dr。Don Bottaro和Marston Linehan)，我们的Grb2信号抑制剂正在细胞研究中进行检测，其中某些药物已被证明可以阻断肝细胞生长因子（HGF）诱导的细胞在含纳摩尔浓度的成纤维细胞Met中的迁移，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，我们的合作者已经证明了在两种侵袭性肿瘤模型中抑制转移，而不影响原发肿瘤的生长速度。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疗效，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：YopH PTP是一种潜在的生物恐怖剂鼠疫耶尔森菌的致病成分，目前正在开发合成的小分子抑制剂。这项工作是与博士合作完成的。罗伯特·乌尔里希（USAMRIID）和大卫·沃（NCI）。使用了一个集中的文库方法，其中两个芳香片段通过一系列连接片段连接在一起。这导致鉴定低微摩尔亲和抑制剂正在进行进一步优化。一种平行的抑制剂开发方法正在进行，该方法依赖于优化YopH底物，为抑制剂的开发提供结构起点。该方法在使用硝基苯基磷酸盐底物方面是独一无二的，它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。最终的抑制剂是通过用水解稳定的生物同工酯取代磷酸酯得到的。这项工作已经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）的关键结合。这项工作涉及到结合Grb2 Src同源3 （SH3）结构域的肽和肽模拟物的合成。闭合环复分解（RCM）已被用于制备大环肽，其在细胞裂解物中表现出增强的阻断同源Grb2-SOS复合物形成的能力。(4) Polo-like Kinase 1 (Plk1) Polo Box结构域结合抑制剂：丝氨酸/苏氨酸Polo-like Kinase 1 （Plk1）的过表达与几种人类癌症的肿瘤发生密切相关。干扰Plk1功能可诱导肿瘤细胞凋亡，但对正常细胞无影响。因此，Plk1是一个潜在的有吸引力的抗癌化疗靶点。Plk1具有独特的磷酸肽结合polo盒结构域（PBD），这对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Plks的四个成员中。因此，它们是选择性抑制Plks功能的理想靶点。通过检查各种PBD结合的磷酸肽，我们的NCI合作者Dr. Kyung Lee先前发现，一个5聚磷酸肽PLHSpT与Plk1 PBD具有高亲和力特异性相互作用，而它不能与两个密切相关的激酶Plk2和Plk3的PBD显著相互作用。从先前报道的含有5个氨基酸的pThr肽开始，通过迭代的顺序过程进行结构优化。与德国莱平大学的Thorsten Berg博士合作测定的Plk1、2和3的PBD结构域的结合亲和力表明，我们已经能够将Plk1 PBD的结合亲和力提高3个数量级以上，同时保持Plk1 PBD的高选择性，而不与相关的Plk2或Plk3 PBD结合。发现了三种不同类型的高亲和结合抑制剂，它们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，解决了这些与Plk1 PBD蛋白结合的肽的x射线共晶结构，显示出一种以前从未观察到的完全意想不到的结合模式。这项工作导致了全新氨基酸类似物的开发，并将其应用于三类选择性高亲和力Plk1 PBD抑制剂。这些抑制剂所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，这将重新定义pbd导向抑制剂的领域。这些化合物可能为一种新型抗癌化学疗法提供基础。