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（由适用提供）：多形胶质母细胞瘤是成年人中最常见的原发性脑肿瘤，也是最致命的恶性肿瘤之一，中位生存期为12-15个月的多种多模态治疗。周围正常大脑的积极入侵使完全的手术切除不可能，增加了对放射线和化学疗法的抵抗力，并实际上确保了肿瘤的复发。这是有很大的未满足的临床需求来开发靶向分散肿瘤细胞以改善该疾病治疗的创新方法。非受体酪氨酸激酶PYK2在收敛的点与来自细胞粘附受体，生长因子受体和G蛋白偶联受体的积分信号传导的功能，这些受体调节细胞增殖，迁移和生存。 PYK2在体外与神经胶质瘤迁移和侵袭密切相关，表达/活性与患者的肿瘤级相关。在鼠胶质母细胞瘤内型型模型模型中，沉默的PYK2表达或PYK2活性的抑制可显着扩展生存率。来自体外和体内模型系统的当前数据支持以下假设：抑制PYK2的生物学活性具有通过限制侵袭并提高对放射和化学疗法的敏感性来改善胶质母细胞瘤患者的临床结果的潜力。酪氨酸激酶抑制剂的临床翻译主要针对竞争性由于对这些结构域的序列和结构的显着保存而缺乏特异性挑战催化结构域的抑制作用。抑制激酶活性的另一种方法是针对调节激酶活性至关重要的靶标。通过介导PYK2低聚，氨基末端FERM结构域在调节PYK2活性中起着核心作用。我们假设FERM结构域代表了治疗的新目标。专门抑制PYK2活性的创新。当前，没有专门针对FERM结构域的小分子。本应用中提出的研究的目的是进行高通量筛选（HTS），以鉴定PYK2的小分子抑制剂作为具有广泛应用的新治疗剂。 PYK2的小分子抑制剂在胶质母细胞瘤，其他侵入性癌症，感染和骨质疏松症中具有治疗性应用。 Alphascreen测定法将充当主要测定法，以鉴定FERM结构域低聚物化的小分子抑制剂。该测定将在基于细胞的测定后检测到全长PYK2激活的细胞内效率。命中证实我将使用基于细胞的FERM二聚化和生化PYK2催化活性测定的IS两个测定。表明在寡聚化测定中效率但在激酶测定中无活跃的效率的命中将使用干粉化合物进行验证，并继续进行神经胶质瘤迁移和入侵评估的功能验证。一项全面的关键路径测试漏斗已经到位，我们预计对其对命中率研究的适用性起点以及对临床前动物模型中未来的体内评估的起点进行了快速评估。