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

异常激酶依赖性信号传导与几种癌症的病因学相关。为此，正在开发药理学试剂以调节激酶依赖性信号传导作为潜在的新抗癌疗法。我们正在开发激酶依赖性信号传导抑制剂，其通过以下方式发挥作用：（1）阻断由polo样激酶1（Plk 1）的polobox结合结构域（PBD）与含磷酸苏氨酸（pThr）/磷酸丝氨酸（pSer）的蛋白质序列的识别和结合介导的蛋白质-蛋白质缔合;（2）阻断细胞蛋白质-酪氨酸磷酸酶（PTP）对磷酰基的去除。丝氨酸/苏氨酸polo样激酶1（Plk 1）的过表达与几种人类癌症的发生密切相关。干扰Plk 1功能可诱导肿瘤细胞凋亡，但不诱导正常细胞凋亡。因此，Plk 1是一个潜在的有吸引力的抗癌化疗靶点。Plk 1具有独特的磷酸肽结合波罗box结构域（PBD），其对于其细胞内定位和有丝分裂功能是必需的。与激酶结构域不同，PBD仅存在于Plks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。通过研究各种PBD结合磷酸肽，我们的NCI合作者Kyung Lee博士先前发现，5-mer磷酸肽PLHSpT以高亲和力特异性地与Plk 1 PBD相互作用，而它不能与两种密切相关的激酶Plk 2和Plk 3的PBD显着相互作用。从该肽开始，我们采用了结构优化的迭代顺序过程来获得新的试剂，其以高亲和力结合Plk 1 PBD。这些试剂中的几种可以在比亲本PLHSpT肽强10，000倍的浓度（低纳摩尔）下抑制与Plk 1 PBD的结合相互作用。这些肽相对于相关Plk 2或Plk 3 PBD保留了对Plk 1 PBD的高选择性。与Michael Yaffe博士（麻省理工学院）合作，这些与Plk 1 PBD结合的肽的X射线共晶体结构表明了利用非配体PBD中不存在的“隐蔽”结合通道的未预料到的结合模式。尽管PBD对肽和蛋白质的高亲和力识别中的关键要素来源于pThr/pSer-残基，但这些残基在治疗中的使用可能受到细胞摄取不良的限制，部分原因是磷酰基部分的高阴离子电荷。我们最近发现了新的合成转化，通过“分子内电荷掩蔽”减少了整体肽的阴离子电荷，这为肽在细胞测定中提供了增强的功效。在进一步的工作中，我们正在开发将抗体的特性与生物活性小分子相结合的蛋白质。这项工作是与Christoph Rader博士（斯克里普斯佛罗里达）合作进行的。我们的方法采用单克隆抗体和抗体Fc片段，其含有单个C-末端硒代半胱氨酸残基（Fc-Sec）。所得抗体药物缀合物（ADC）通过改变它们所缀合的肽或小分子而针对多种靶标。在我们工作的一个方面，我们已经采用了各种化学物质来连接生物可切割的接头，一旦实现递送至靶标，所述接头就允许释放货物。我们已经开发了多功能的异双功能接头，其结合了与多种类型的无Cu Huisgen 1，3-偶极环加成试剂相容的生物可裂解键。这些接头含有靶向功能和药物有效载荷。在我们工作的一个方面，涉及有效的细胞毒性肽，单甲基澳瑞他汀F（MMAF），我们正在研究可以通过亲核烷基化反应与Fc-Sec蛋白缀合的接头。这项工作涉及开发MMAF肽关键组分的新合成路线。

Abberant 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. We are developing kinase-dependent signaling inhibitors that function by: (1) Blocking protein-protein associations mediated by recognition and binding of the polobox binding domain (PBD) of polo-like kinase 1 (Plk1) to phosphothreonine (pThr)/phosphoserine (pSer)-containing protein sequences and (2) Blocking the removal of phosphoryl groups by cellular protein-tyrosine phosphatases (PTPs). 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 phosphopeptide binding 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 the 5-mer 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 this peptide, we employed an iterative sequential process of structural refinement to arrive at new agents, which bind with high affinity to the Plk1 PBD. Several of these agents can inhibit binding interactions with the Plk1 PBD at concenterations (low nanomolar) that are 10,000-times more potent than the parent PLHSpT peptide. These peptides retain high selectivity for the Plk1 PBD relative to the related Plk2 or Plk3 PBDs. In collaboration with Dr. Michael Yaffe (MIT) X-ray co-crystal structures of these peptides bound to Plk1 PBD indicate unanticipated modes of binding that take advantage of a "cryptic" binding channel that is not present in the non-liganded PBD. Although critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues, the use of these residues in therapeutics is potentially limited by poor cellular uptake, in part due to high anionic charge of the phosphoryl moiety. We have recently discovered new synthetic transformations that lessen the overall peptide anionic charge by "intramolecular charge masking," which provides peptides with enhanced efficacy in cellular assays. In further work we are developing proteins that merge properties of antibodies with biologically active small molecules. This work is being done in collaboration with Dr. Christoph Rader (Scripps Florida). Our approach employs monoclonal antibodies and antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec). The resulting antibody drug conjugates (ADCs) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In one aspect of our work, we have employed a variety of chemistries to attach biologically-cleavable linkers that allow release of cargo once delivery to the target has been achieved. We have developed versatile hetero-bifunctional linkers incorporating biologically cleavable bonds that are compatible with multiple types of Cu-free Huisgen 1,3-dipolar cycloaddition reagents. These linkers contain both targeting functionality and drug payloads. In one aspect of our work involving the potently cytotoxic peptide, monomethyl auristatin F (MMAF), we are examining linkers that can be conjugated to the Fc-Sec protein by nucleophilic alkylation reactions. This work has involved developing new synthetic routes to key components of the MMAF peptide.