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

靶向蛋白激酶(PK)依赖的信号转导：异常的PK依赖信号转导与几种癌症的病因有关。出于这个原因，正在开发药理学药物来调节依赖于激酶的信号，作为潜在的新的抗癌治疗药物。我们正在开发依赖PK的信号抑制剂，目标是四个关键成分：(1)通过识别和结合src同源2(SH2)结构域与磷酸酪氨酰(PTyr)残基介导的蛋白质-蛋白质关联；(2)通过细胞蛋白-酪氨酸磷酸酶(PTPs)去除pTyr磷酸基。(3)Src同源3(SH3)结构域介导的蛋白质-蛋白质结合；(4)Polo-like kinase1的Polobox结合结构域；(1)SH2结构域结合抑制物。高亲和力生长因子受体结合蛋白2(Grb2)结合拮抗剂正在被制备成治疗erbB-2和c-Met依赖的癌症的潜在新疗法。作为与NCI临床研究人员(Don Bottaro博士和Marston Linehan博士)合作工作的一部分，我们的Grb2信号抑制剂正在进行细胞研究，其中某些药物已被证明可以纳摩尔浓度阻止肝细胞生长因子(HGF)诱导的蛋氨酸成纤维细胞迁移，并抑制可能参与血管生成的小管形成。使用我们的一种制剂，我们的合作者已经在两个侵袭性肿瘤模型中展示了抑制体内转移的作用，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：正在开发针对YopH PTP的合成小分子抑制剂，YopH PTP是潜在的生物毒剂鼠疫耶尔森氏菌的致病成分。这项工作是与罗伯特·乌尔里希博士(USAMRIID)和大卫·沃(NCI)合作完成的。已经使用了聚焦文库方法，其中两个芳香族片段通过一系列接头片段连接在一起。这导致了正在进行进一步优化的低微摩尔亲和力抑制剂的识别。目前正在进行一种平行的缓蚀剂开发方法，依赖于YopH底物的优化，为缓蚀剂开发提供结构起点。该方法的独特之处在于它使用了硝基苯磷酸底物，允许通过简单地测量黄色衍生反应产物硝基苯酚来监测底物的释放。最终的抑制剂是通过用可水解稳定的生物异构体取代磷酸酯来获得的。这项工作已经产生了低纳摩尔的非混杂抑制剂。(3)SH3结构域结合抑制剂。我们已经承担了基于多肽的抑制剂的开发，以阻断Grb2与其结构性结合伙伴SOS(SOS)的关键结合。这项工作涉及到与Grb2Src同源3(SH3)结构域结合的多肽和多肽模拟物的合成。闭环歧化反应(RCM)已被用于制备大环肽，这种大环肽具有更强的阻断细胞裂解产物中同源Grb2-SOS复合体形成的能力。(4)Polo-like Kinase 1(Plk1)Polo Box结构域结合抑制物：丝氨酸/苏氨酸Polo-like kinase1(Plk1)的过表达与几种人类癌症的发生密切相关。干扰Plk1功能可诱导肿瘤细胞的凋亡，但不能诱导正常细胞的凋亡。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的Polo盒结构域(PBD)，这是其细胞内定位和有丝分裂功能所必需的。与激酶结构域不同，PBD只在Plk的四个成员中发现。因此，它们是选择性抑制Plks功能的理想靶点。通过研究各种与PBD结合的磷酸肽，我们的NCI合作者Kyung Lee博士先前发现，5聚体磷酸肽PLHSpT与Plk1 PBD具有高亲和力，而它无法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多肽开始，经过结构优化的迭代序列过程。与德国莱普津大学的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，我们能够将Plk1 PBD的结合亲和力提高3个数量级以上，同时保持对Plk1 PBD的高选择性，而不与相关的PLK2或Plk3 PBD结合。发现了三类截然不同的高亲和力结合抑制剂，它们含有新的、迄今未见报道的氨基酸类似物。在与Michael Yaffe(麻省理工学院)博士的合作下，这些多肽与Plk1 PBD蛋白结合的X射线共晶结构被解开，揭示了一种以前从未观察到的完全意想不到的结合模式。这项工作导致了全新氨基酸类似物的开发，并将它们应用于三类选择性的、高亲和力的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，这将重新定义PBD导向的抑制剂领域。这些化合物可能为新型抗癌化疗药物的开发奠定基础。

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.