HTS for Identification of Novel Inhibitors of Pyk2 Activity

HTS 用于鉴定 Pyk2 活性的新型抑制剂

基本信息

批准号：
9245558
负责人：
JOSEPH C LOFTUS
金额：
$ 40.63万
依托单位：
MAYO CLINIC ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9245558
关键词：
Adhesions Adjuvant Adult Adverse effects Alpha Cell Animal Model Apoptotic Basic Science Binding Biochemical Biological Biological Assay Biophysics Brain Catalytic Domain Cell Proliferation Cells Central Nervous System Neoplasms Cessation of life Chemicals Clinical Clinical Treatment Complement Complex Critical Pathways Data Databases Development Dimerization Disease Effectiveness Evaluation Excision Future G-Protein-Coupled Receptors Glioblastoma Glioma Goals Growth Growth Factor Receptors In Vitro Inflammation Integrins Invaded Investigation Lead Length Libraries Luciferases Malignant Glioma Malignant Neoplasms Mediating Modality Molecular Molecular Target Monoclonal Antibodies Mus Mutation N-terminal Operative Surgical Procedures Osteoporosis Outcome Pathway interactions Patients Pharmaceutical Preparations Phosphotransferases Play Positioning Attribute Powder dose form Pre-Clinical Model Primary Brain Neoplasms Promega PubChem Radiation Reactive Inhibition Recombinants Recurrence Regimen Regulation Reporter Resistance Role Signal Transduction Specificity Structure Support System Surface Testing Therapeutic Therapeutic Agents Tyrosine Kinase Inhibitor Validation Vitronectin Xenograft Model Xenograft procedure adhesion receptor base cancer cell cancer invasiveness cell motility chemotherapy clinical translation cross reactivity dimer drug development experience ezrin high throughput screening improved improved outcome in vivo in vivo Model inhibitor/antagonist innovation insight knock-down method development migration miniaturize moesin neoplastic cell new therapeutic target novel novel therapeutics pre-clinical programs protein protein interaction public health relevance receptor scale up screening small molecule small molecule inhibitor statistics temozolomide therapeutic target tumor virtual

项目摘要

DESCRIPTION (provided by applicant): Glioblastoma multiforme is the most frequent primary brain tumor in adults and one of the most lethal malignancies with a median survival of 12-15 months despite multi-modality treatment. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and virtually assures tumor recurrence. Thus, there is a significant unmet clinical need to develop innovative approaches to target the dispersing tumor cells for improved treatment of this disease. The non-receptor tyrosine kinase Pyk2 functions at a point of convergence to integrate signaling from cell adhesion receptors, growth factor receptors, and G protein coupled receptors that regulate cell proliferation, migration, and survival. Pyk2 is intimately involved in glioma migration and invasion in vitro and increased expression/activity strongly correlates with advancing tumor grade in patients. Silencing Pyk2 expression or inhibition of Pyk2 activity significantly extends survival in a murine glioblastoma intracranial xenograft model. Current data from both in vitro and in vivo model systems support the hypothesis that inhibition of the biological activities of Pyk2 has potential to improve the clinicl outcome of glioblastoma patients by limiting invasion and increasing sensitivity to radiation and chemotherapy. Clinical translation of tyrosine kinase inhibitors is largely directed at competitive inhibition of catalytic domains which is challenged by lack of specificity due to the significant conservation of both sequence and structure of these domains. An alternative approach to the inhibition of kinase activity is to target domains essential for the regulation of kinase activity.By mediating Pyk2 oligomerization, the amino terminal FERM domain plays a central role in the regulation of Pyk2 activity. We hypothesize that the FERM domain represents a novel target for therapeutic innovation to specifically inhibit Pyk2 activity. Currently, there are no small molecules that specifically target FERM domains. The goal of the studies proposed in this application is to conduct a high throughput screen (HTS) for the identification of small-molecule inhibitors of Pyk2 as new therapeutic agents with wide applicability. Small molecule inhibitors of Pyk2 have therapeutic application in glioblastoma, other invasive cancers, inflammation, and osteoporosis. An AlphaScreen assay will function as primary assay to identify small-molecule inhibitors of FERM domain oligomerization. This assay will be followed by cell-based assays detecting intracellular efficacy of inhibition of full-length Pyk2 activation. The hits confirmed i both assays will be prioritized using cell-based FERM dimerization and biochemical Pyk2 catalytic activity assays. The hits demonstrating efficacy in the oligomerization assay, yet inactive in kinase assay, will be validated using dry powder compounds and proceed to functional validation in glioma migration and invasion assays. A comprehensive critical path testing funnel is already in place and we anticipate rapid evaluation of hits for their suitabilityas starting points for hit-to-lead studies and for future in vivo evaluation in pre-clinical animal models.

描述（由申请人提供）：多形性胶质母细胞瘤是成人中最常见的原发性脑肿瘤，也是最致命的恶性肿瘤之一，尽管采用多种治疗方法，但其中位生存期仍为 12-15 个月。手术切除是不可能的，增加了对放疗和化疗的抵抗力，并且实际上确保了肿瘤复发，因此，开发针对分散的肿瘤细胞以改善这种疾病的治疗的创新方法存在显着的未满足的临床需求。非受体酪氨酸激酶 Pyk2 在汇聚点发挥作用，整合来自细胞粘附受体、生长因子受体和 G 蛋白偶联受体的信号传导，调节细胞增殖、迁移和存活。 Pyk2 与神经胶质瘤体外迁移和侵袭密切相关。表达/活性的增加与患者肿瘤分级的进展密切相关，沉默 Pyk2 表达或抑制 Pyk2 活性可显着延长小鼠颅内胶质母细胞瘤的生存期。异种移植模型。来自体外和体内模型系统的当前数据支持这样的假设：抑制 Pyk2 的生物活性有可能通过限制侵袭和增加对酪氨酸的临床转化来改善胶质母细胞瘤患者的临床结果。激酶抑制剂主要针对竞争性催化结构域的抑制由于缺乏特异性而受到挑战，因为这些结构域的序列和结构都显着保守。抑制激酶活性的另一种方法是针对激酶活性调节所必需的结构域。通过介导 Pyk2 寡聚化。，氨基末端 FERM 结构域在 Pyk2 活性的调节中发挥着核心作用，我们认为 FERM 结构域代表了特异性抑制 Pyk2 活性的治疗创新的新靶标。本申请提出的研究的目标是进行高通量筛选（HTS），以鉴定 Pyk2 小分子抑制剂作为具有广泛适用性的新治疗剂。、其他侵袭性癌症、炎症和骨质疏松症将作为主要检测来识别 FERM 结构域寡聚化的小分子抑制剂。检测全长 Pyk2 激活的细胞内抑制功效的基于细胞的测定将优先使用基于细胞的 FERM 二聚化和生化 Pyk2 催化活性测定。激酶测定，将使用干粉化合物进行验证，并继续在神经胶质瘤迁移和侵袭测定中进行功能验证，一个全面的关键路径测试漏斗已经就位，我们预计对其命中进行快速评估。适用性作为先导化合物研究和未来临床前动物模型体内评估的起点。