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

异常的激酶依赖性信号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。激酶依赖性信号传导涉及三个关键组成部分：(1)关键细胞蛋白中磷酸化氨基酸残基的产生；(2)其他信号蛋白对这些残基的识别和结合；(3)细胞磷酸酶去除氨基酸磷酸化基团。对于蛋白酪氨酸激酶（PTKs），这三个组成部分包括：(1)PTKs产生含磷酸酪氨酸（pTyr）的序列；(2)通过src同源性2 （SH2）结构域识别和结合pTyr序列；(3)蛋白酪氨酸磷酸酶（PTPs）通过磷酸酯水解破坏pTyr序列。因此，这个项目的一个统一主题是设计和合成针对这三种成分的抑制剂。在SH2结构域，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。最近，我们采用了一种免费的方法来阻断不靶向SH2结构域的Grb2功能。在这种方法中，正在开发抑制剂，阻断Grb2与其组成性结合伙伴，七子之子（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。人们一直在努力开发SH2结构域导向的shc依赖性信号的肽模拟抑制剂。Shc蛋白是一种非催化SH2结构域对接模块，参与多种与增殖、存活和凋亡相关的细胞调控过程。Shc和Grb2蛋白对于PTKs的下游信号传导尤其重要，它们已被证明将细胞质激酶结构域的激活与Ras效应器联系起来。Shc也被证明是c-Met和ErbB2 RTK癌蛋白下游血管内皮生长因子（VEGF）产生的关键血管生成开关，其中Shc而不是Grb2的募集已被证明是必需的事件。因此，通过阻断其SH2结构域来破坏shc依赖性信号可能为依赖此类PTKs失调的癌症提供新的治疗方法。先前有报道称，14-mer的ζ链t细胞受体肽Ac-GHDGLpYQGLSTATK-amide（其中pY = pTyr）与Shc SH2结构域结合，亲和力为Kd = 50 &#956；M。我们制备了含有不存在于遗传编码氨基酸的功能的类似物，包括n -烷基甘氨酸（肽）残基。这项工作的结果是发现了具有良好Shc SH2结构域结合亲和力的四聚体肽类杂交种。这些药物的进一步结构优化正在进行中。为了进行基于细胞的研究，膜载体肽序列被化学连接到这些努力产生的高亲和力磷酸肽。初步数据表明，这些肽可以在低微摩尔浓度下阻断Shc与活化Met在全细胞中的结合。丝氨酸/苏氨酸polo样激酶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。利用我们实验室开发的独特的“后固相多样化”技术，我们优化了一系列肽和肽模拟物的结合亲和力。这导致PBD1结合亲和力提高了100倍以上。Michael Yaffe博士（麻省理工学院）的实验室正在研究与PBD1蛋白结合的高亲和力配体的x射线晶体结构。这些研究结果将有助于进一步优化pbd1结合抑制剂的结构。在磷酸酶领域，正在开发针对YopH PTP的抑制剂，YopH PTP是潜在的生物恐怖剂鼠疫耶尔森菌的致病成分。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中。该方法的不同寻常之处在于，它依赖于YopH底物的优化，为抑制剂的开发提供结构起点。最终的抑制剂将通过水解稳定的生物同工酯取代磷酸酯来获得。在PTK抑制剂开发领域，高亲和力的Sugen Pharmaceuticals- c-Met激酶抑制剂SU11274正被用作结构阐述的起点。x射线晶体研究表明，SU11274的c-Met结合相互作用被很好地限制在催化间隙内。我们在SU11274核心上人工添加了连接子链，以保留催化裂孔内原有的结合，同时在催化裂孔外引入额外的结合相互作用。这项工作有可能推动PTK抑制剂的发展。

Aberrant kinase-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. Kinase-dependent signaling involves three critical components: (1) The generation of phosphorylated amino acid residues in key cellular proteins; (2) the recognition and binding to these residues by other signaling proteins and (3) the removal of the amino acid phosphoryl group by cellular phosphatases. For protein-tyrosine kinases (PTKs) these three components consist of: (1) The generation of phosphotyrosyl (pTyr)-containing sequences by PTKs; (2) The recognition and binding to pTyr-containing sequences by src homology 2 (SH2) domains and (3) The destruction of pTyr sequences through phosphate ester hydrolysis by protein tyrosine phosphatases (PTPs). Accordingly, a unifying theme of this project is the design and synthesis of inhibitors directed at each of these three components. In the SH2 domain area, 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. More recently, we have undertaken a complimentary approach to blocking Grb2 function that does not target the SH2 domain. In this approach inhibitors are being developed 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. Efforts have been been undertaken to develop SH2 domain-directed peptide mimetic inhibitors of Shc-dependent signaling. Shc proteins are non-catalytic SH2 domain-containing docking modules that participate in a variety of cell-regulatory processes associated with proliferation, survival and apoptosis. Shc as well as Grb2 proteins are particularly important for down stream signaling of PTKs, where they have been shown to link activation of the cytoplasmic kinase domains with Ras effectors. Shc has also been shown to serve as a critical angiogenic switch for for the production of vascular endothelial growth factor (VEGF) downstream from the c-Met and ErbB2 RTK oncoproteins, where recruitment of Shc but not Grb2 has been shown to be a required event. Accordingly, disruption of Shc-dependent signaling through blockade of its SH2 domain may afford a new therapeutic approach to cancers reliant on disregulation of such PTKs. It has previously been reported that the 14-mer zeta-chain-T cell receptor peptide, Ac-GHDGLpYQGLSTATK-amide (where pY = pTyr) binds to the Shc SH2 domain with an affinity of Kd = 50 μM. We prepared analogues that contained functionality not present in genetically-encoded amino acids, including N-alkylglycine (peptoid) residues. This work resulted in the discovery of tetrameric peptide peptoid hybrids that exhibit good Shc SH2 domain binding affinity. Further structural optimization of these agents is in progress. In order to conduct cell-based studies, membrane carrier peptide sequences were chemically linked to select high affinity phosphopeptides resulting from these efforts. Preliminary data has indicated that these peptides can block the binding of Shc to activated Met in whole cells at low micromolar concentrations. 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. Using a unique "post solid-phase diversification" technique developed in our laboratory, we have optimized the binding affinity of a series of peptides and peptide mimetics. This has resulted in greater than 100-fold enhancement in PBD1 binding affinity. Work is in progress in the laboratory of Dr. Michael Yaffe (MIT) to solve the X-ray crystal structure of our high affinity ligands bound to PBD1 protein. The results of these studies should facilitate a further structure-based optimization of our PBD1-binding inhibitors. In the phosphatase area, 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. The approach is unusual in that it relies on the optimization of YopH substrates to provide structural starting points for inhibitor development. Final inhibitors will be obtained by replacing the phosphate esters by hydrolytically stable bioisosteres. In the area of PTK inhibitor development, the high affinity Sugen Pharmaceuticals- c-Met kinase inhibitor SU11274 is being used as a starting point for structural elaboration. X-ray crystal studies have shown that c-Met binding interactions of SU11274 are confined well within the catalytic cleft. We have synthetically appended linker chains onto the SU11274 core to retain the original binding within the catalytic cleft while at the same time introduction additional binding interactions exterior to catalytic cleft. This work has the potential to advance the development of PTK inhibitors in general.