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

酪氨酸激酶依赖性信号传导抑制剂作为抗癌药物

• 批准号: 8552595
• 负责人: TERRENCE BURKE
• 金额: $ 93.18万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552595
• 关键词: Affinity; Alkylation; Amino Acid Sequence; Amino Acids; Antibodies; Antineoplastic Agents; Apoptosis; Binding; Biological Availability; Bioterrorism; C-terminal; CCR; Charge; Chemistry; Collaborations; Cytotoxic agent; Development; Etiology; Excision; Exhibits; F-peptide; Fc Immunoglobulins; Germany; Human; Immunoglobulin Fragments; Indium; Libraries; Ligands; Light; Malignant Neoplasms; Mediating; Mitotic; Modification; New Agents; Normal Cell; Peptide Sequence Determination; Peptides; Pharmaceutical Preparations; Phosphoamino Acids; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphoserine; Phosphothreonine; Phosphotransferases; Plague; Polo-Box Domain; Process; Property; Protein Binding Domain; Protein Kinase; Protein Tyrosine Phosphatase; Proteins; Reaction; Reagent; Relative (related person); Roentgen Rays; Route; Selenocysteine; Series; Serine; Signal Transduction; Structure-Activity Relationship; Therapeutic; Threonine; Tyrosine Kinase Inhibitor; Universities; Work; Yersinia pestis; Yersinia yopH protein; analog; anti-cancer therapeutic; cytotoxic; design; human PLK1 protein; inhibitor/antagonist; member; mimetics; neoplastic cell; novel; overexpression; peptide structure; phosphatase inhibitor; small molecule; tumorigenesis; uptake

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 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 phosphotyrosyl (pTyr) 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 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 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. In collaboration with Dr. Thorsten Berg (The University of Leipzing, Germany), we have been able to show that the Plk1 PBD binding affinity is increased by over 3 - orders of magnitude, while retaining high selectivity for the Plk1 PBD relative to the related Plk2 or Plk3 PBDs. Three distinct classes of high affinity-binding inhibitors were discovered, which contain new 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. These reveal an unanticipated mode of binding. The work resulted in the development of new amino acid analogues and their application to three classes of selective, high affinity Plk1 PBD inhibitors. 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/pSer residues, which form key components of ligand recognition. 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 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/pSer replacements within a PBD context. Accordingly, we have examined the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements. This work has shed new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.(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 (CCR, 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 development of a low-nanomolar non-promiscuous inhibitors that showed significant inhibition of intracellular Y. pestis replication at a non-cytotoxic concentration. 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 (CCR, NCI). Our approach employs antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec), which 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 are employing click chemistry 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 that are compatible with multiple types of Cu-free click reagents and that incorporate biologically cleavable bonds. 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, which 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.

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，其功能是：(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)对磷酸酪氨酰(PTyr)磷酸基的去除。(1)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)的过度表达与几种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。在与德国莱普津大学Thorsten Berg博士的合作下，我们已经能够证明Plk1 PBD的结合亲和力提高了3个数量级以上，同时相对于相关的PLK2或Plk3 PBD保持了对Plk1 PBD的高选择性。发现了三类不同的高亲和力结合抑制剂，它们含有新的氨基酸类似物。在与Michael Yaffe(麻省理工学院)博士的合作下，解决了这些与Plk1 PBD蛋白结合的多肽的X射线共晶结构。这些揭示了一种意想不到的约束模式。这项工作导致了新的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结合模式定义了一种新的PBD结合相互作用类型，这可能会极大地影响PBD导向的抑制剂领域。进一步的工作是对形成配体识别关键成分的pThr/pSer残基进行修饰。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它们在细胞磷酸酶存在下的不稳定性以及由于它们的高阴离子电荷而导致的细胞摄取不足的限制。到目前为止，很少有关于pThr/pSer替换在PBD上下文中的研究。因此，我们研究了各种氨基酸残基和衍生物作为pThr/pSer替代品的能力。这项工作为PBD识别磷酸氨基酸模拟物的构效关系提供了新的线索。(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(CCR，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。这导致了一种低纳摩尔非混杂抑制剂的开发，该抑制剂在非细胞毒性浓度下对鼠疫杆菌的细胞内复制显示出显著的抑制作用。为了补充这项工作，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(CCR，NCI)合作完成的。我们的方法使用含有单一C-末端硒半胱氨酸残基(FC-SEC)的抗体Fc片段，通过改变它们所结合的肽或小分子来针对不同的靶标。在我们工作的一个方面，我们正在使用点击化学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，它与多种类型的无铜点击试剂兼容，并结合了生物可切割的键。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)，我们正在检查可生物切割的连接体，它可以通过亲核烷基化反应连接到FC-SEC蛋白上。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。