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

Abberant激酶依赖信号与几种癌症的病因学有关。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。我们正在开发激酶依赖的信号抑制药，其功能如下：(1)通过识别和结合Polo-like kinase1(Plk1)的Polobox结合域(PBD)与含有磷苏氨酸(PThr)/磷酸丝氨酸(PSer)的蛋白质序列而介导的蛋白质-蛋白质关联；(2)阻断细胞蛋白-酪氨酸磷酸酶(PTPs)对磷酸基的去除。丝氨酸/苏氨酸Polo-like kinase1(Plk1)的过度表达与多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具有一个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。从这一肽开始，我们采用了结构精化的迭代序列过程来获得与Plk1 PBD具有高亲和力的新试剂。其中几种药物可以抑制与Plk1 PBD的结合作用，其浓度(低纳摩尔)比亲本PLHSpT肽的效力高10,000倍。相对于相关的PLK2或Plk3 PBD，这些多肽对Plk1 PBD保持了较高的选择性。在与Michael Yaffe(麻省理工学院)博士的合作中，这些与Plk1 PBD结合的多肽的X射线共晶结构表明，这些多肽利用了非配体PBD中不存在的“隐蔽”结合通道，具有意想不到的结合模式。尽管PBD对多肽和蛋白质的高亲和力识别的关键元件来自pThr/pSer-残基，但这些残基在治疗中的使用可能受到细胞摄取能力差的限制，部分原因是磷酸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”减少了整体多肽的阴离子电荷，这为多肽在细胞分析中提供了更好的效果。在进一步的工作中，我们正在开发融合抗体特性和生物活性小分子的蛋白质。这项工作是与克里斯托夫·雷德博士(佛罗里达州斯克里普斯)合作完成的。我们的方法使用了含有单一C末端硒半胱氨酸残基的单抗和抗体Fc片段(Fc-SEC)。由此产生的抗体药物结合物(ADC)通过改变它们所结合的多肽或小分子来针对各种靶点。在我们工作的一个方面，我们使用了各种化学物质来连接生物可切割的连接物，一旦实现了对目标的交付，就允许释放货物。我们已经开发了多功能的异双功能连接物，含有生物可切割的键，与多种类型的无铜惠氏1，3-偶极环加成试剂兼容。这些链接器同时包含靶向功能和药物有效载荷。在我们工作的一个方面，涉及到有效的细胞毒肽，单甲基金黄色素F(MMAF)，我们正在检查可以通过亲核烷基化反应连接到FC-SEC蛋白上的连接体。这项工作包括开发新的合成路线，以获得MMAF多肽的关键成分。

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.