Inhibitors of Tyrosine Kinase-Dependent Signaling 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。我们正在开发激酶依赖性信号抑制剂，其功能如下：(1)阻断由polo样激酶1 （Plk1）的polobox结合域（PBD）与含磷苏氨酸（pThr）和磷丝氨酸（pSer）的蛋白序列识别和结合介导的蛋白-蛋白关联；(2)阻断细胞蛋白酪氨酸磷酸酶（PTPs）去除磷酸化基团。(1) 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盒子结构域（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，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。从这个肽开始，我们采用了一个迭代的顺序结构优化过程来获得新的药物，这些药物与Plk1 PBD具有高亲和力。其中一些药物可以抑制与Plk1 PBD结合的相互作用，其浓度（低纳摩尔）比亲本PLHSpT肽强10000倍。相对于相关的Plk2或Plk3 PBD，这些肽对Plk1 PBD保持高选择性。在与Michael Yaffe博士（麻省理工学院）的合作中，这些与Plk1 PBD结合的肽的x射线共晶结构表明了意想不到的结合模式，这些模式利用了非配体PBD中不存在的隐结合通道。在进一步的工作中，我们研究了一系列氨基酸类似物，旨在利用新发现的隐结合通道。与亲本PLHSpT肽相比，这些氨基酸类似物中的一些表现出显著增加的抑制效力。这些抑制剂表现出的结合模式定义了一种新的pbd结合相互作用类型，可以极大地影响pbd定向抑制剂领域。进一步的工作是针对pThr残基的修饰，它是配体识别的关键组成部分。虽然PBD对多肽和蛋白质的高亲和力识别的关键元素来源于pThr或pSer残基，但这些残基在细胞磷酸酶存在下的不稳定性以及由于其高阴离子电荷而导致的细胞摄取不良，可能限制了它们在治疗中的使用。迄今为止，在PBD的背景下，很少有关于pThr或pSer替代的研究。因此，我们研究了各种氨基酸残基和衍生物作为pThr或pSer替代品的能力。这项工作为磷酸氨基酸模拟物PBD识别的结构活性关系提供了新的思路。特别值得注意的是，我们发现单阴离子pThr类似物与Plk1 PBD结合具有个位数抑制效力，并且在细胞分析中表现出增强的功效。我们通过应用生物可逆磷酸基药物前保护扩展了这些发现，在细胞分析中产生了具有进一步增强效力的不带电物种。(2) PTP抑制剂：鉴于蛋白酪氨酸磷酸酶（PTP）家族在癌症中的重要性，我们迄今为止的工作主要集中在蛋白酪氨酸磷酸酶（PTP）家族上，我们正在开始研究双特异性磷酸酶（DUSPs）家族的结构生物学，该家族可以水解pTyr和pThr或含有pser的底物。这项工作使用了一组由我们的合作者组装的磷酸酶。沃和乌尔里希。该图中的酶包括VH1（与引起天花的天花病毒有关）；VHR（牛痘h1相关，在几种癌症中上调）；DUSP12（葡萄糖激酶相关双特异性磷酸酶）；DUSP14;DUSP22;DUSP27， CDC25C和PRL-3（也称为PTP4A3）。选择大多数DUSPS是因为它们与肿瘤有关。Waugh博士正在对结构进行x射线晶体学测定，对于一些DUSP家族成员，这些将是第一次报道的结构。我们正在研究影响不同底物对每一种磷酸酶亲和力的因素，使用栓系肟库方法。我们已经在各种上下文中演示了这种方法的实用性。从亲本肽序列开始，我们在n端生物素化亲本肽的每个位置依次引入一个含氨基的残基，该亲本肽序列在几个磷酸酶家族成员之间表现出良好的亲和力。评估每个残基位置的肟基文库代表了一种栓系片段方法，它允许探索结构多样性，这远远超出了使用编码氨基酸所能达到的。在Ulrich博士的实验室进行的工作中，我们对含肟肽库进行了筛选，以确定其对一系列磷酸酶的底物活性。利用微阵列技术，将含肟肽打印到含亲和素的载玻片（FAST载玻片）上，实现了相对底物效率的测定。每张幻灯片包含大约1000个多肽。打印的磷酸肽阵列被每一组磷酸酶水解，剩余的未水解肽的相对水平用ELISA技术可视化。预计这些研究的数据将有助于设计针对磷酸酶面板成员的肽模拟抑制剂。最后，为了补充这项工作，我们正在开发将抗体特性与生物活性小分子结合在一起的蛋白质。这项工作是与Christoph Rader博士（佛罗里达州Scripps）合作完成的。我们的方法采用单克隆抗体和含有单个c端硒代半胱氨酸残基（Fc- sec）的抗体Fc片段。由此产生的抗体药物偶联物（adc）通过改变其偶联的肽或小分子来针对各种靶标。在我们工作的一个方面，我们使用了多种化学物质来附着生物可切割的连接物，一旦到达目标，就可以释放货物。我们已经开发了多用途的杂双功能连接剂，结合生物可切割的键，与多种类型的无cu Huisgen 1,3-偶极环加成试剂兼容。这些连接器包含靶向功能和药物有效载荷。在我们的工作的一个方面涉及到强细胞毒性肽，单甲基auristatin F (MMAF)，我们正在研究可以通过亲核烷基化反应偶联到Fc-Sec蛋白的生物可切割连接物。这项工作包括开发新的合成途径，以获得MMAF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) and phosphoserine (pSer)-containing protein sequences and (2) Blocking the removal of phosphoryl groups by cellular protein-tyrosine phosphatases (PTPs). (1) 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 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 concentrations (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 cocrystal 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 nonliganded PBD. In further work, we have examined a range of amino acid analogs designed to take advantage of the newly discovered cryptic binding channel. Several of these amino acid analogs exhibit significantly increased inhibitory potencies relative to the parent PLHSpT peptide. The binding modes exhibited by these inhibitors define a new genre of PBD-binding interactions that could greatly impact the field of PBD-directed inhibitors. Further work has been directed at modification of the pThr residue, which forms a key component of ligand recognition. Although critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr or pSer residues, the use of these residues in therapeutics is potentially limited by their lability in the presence of cellular phosphatases and by their poor cellular uptake due to their high anionic charge. To date, there has been little examination of pThr or pSer replacements within a PBD context. Accordingly, we have investigated the abilities of a variety of amino acid residues and derivatives to serve as pThr or pSer replacements. This work has shed new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics. Of particular note, is our discovery of mono-anionic pThr analogues that bind to the Plk1 PBD with single digit inhibitory potencies, and which exhibit enhanced efficacies in cellular assays. We have extended these findings by applying bio-reversible phosphoryl prodrug protection that yielded uncharged species with further enhanced potencies in cellular assays. (2) PTP Inhibitors: While our work to date has focused on the protein-tyrosine phosphatase (PTP) family, given their importance in cancer, we are initiating work to investigate the structural biology of a family of dual specificity phosphatases (DUSPs), which hydrolyze both pTyr and pThr or pSer-containing substrates. . This work employs a panel of phosphatases assembled by our collaborators, Drs. Waugh and Ulrich. Among the enzymes in this panel are VH1 (associated with the Variola virus, which causes small pox); VHR (Vaccinia H1-related, which is upregulated in several cancers); DUSP12 (glucokinase-associated dual specificity phosphatase); DUSP14; DUSP22; DUSP27, CDC25C and PRL-3 (also known as PTP4A3). The majority of the DUSPS were chosen for their oncogenic connections. X-ray crystallographic determination of structures is being undertaken by Dr. Waugh, and for several of the DUSP family members, these will represent the first reported structures. We are investigating factors that influence differential substrate affinity for each phosphatase using a tethered oxime library approach. We have demonstrated the utility of this approach in various contexts. Starting from a parent peptide sequence, which shows good affinity across several members of the family of phosphatases, we sequentially introduced an aminooxy-containing residue at every position of an N-terminally biotinylated parent peptide. Evaluating libraries of oximes at each residue position represents a tethered fragment approach, which allows exploration of structural diversity significantly beyond what would be attainable using coded amino acids. In work performed in the laboratory of Dr. Ulrich, our libraries of oxime-containing peptides were screened for their substrate activities against the panel of phosphatases. The determination of relative substrate efficacies was achieved using microarray techniques involving printing of the oxime-containing peptides onto avidin-containing slides (FAST slides). Each slide contained approximately 1000 peptides. The printed phosphopeptide arrays were subjected to phosphatase hydrolysis by each member of the panel of phosphatases, and the relative levels of non-hydrolyzed peptides remaining were then visualized using ELISA techniques. It is anticipated that data from these studies should facilitate the design of peptide mimetic inhibitors directed against members of the phosphatase panel. Finally, to compliment this 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 bio-cleavable 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.