HTS for Identification of Novel Inhibitors of Pyk2 Activity

 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，尽管采用多种方式治疗，中位生存期为12-15个月。周围正常大脑的侵袭性侵袭使得完全的手术切除变得不可能，增加了对放射和化学疗法的抵抗力，并且实际上确保了肿瘤复发。因此，存在显著的未满足的临床需求，以开发靶向分散的肿瘤细胞的创新方法，用于改善该疾病的治疗。非受体酪氨酸激酶Pyk 2在会聚点处起作用以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，所述细胞粘附受体、生长因子受体和G蛋白偶联受体调节细胞增殖、迁移和存活。Pyk 2在体外与胶质瘤迁移和侵袭密切相关，并且表达/活性增加与患者中肿瘤分级的进展密切相关。沉默Pyk 2表达或抑制Pyk 2活性显著延长了鼠胶质母细胞瘤颅内异种移植模型的存活期。目前来自体外和体内模型系统的数据支持以下假设：抑制Pyk 2的生物活性具有通过限制侵袭和增加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是针对竞争性的 催化结构域的抑制受到由于这些结构域的序列和结构的显著保守而缺乏特异性的挑战。另一种抑制激酶活性的方法是靶向调节激酶活性所必需的结构域，通过介导Pyk 2寡聚化，氨基末端FERM结构域在Pyk 2活性的调节中起核心作用。我们假设FERM结构域代表了特异性抑制Pyk 2活性的治疗创新的新靶点。目前，还没有特异性靶向FERM结构域的小分子。本申请中提出的研究的目标是进行高通量筛选（HTS）以鉴定Pyk 2的小分子抑制剂作为具有广泛适用性的新治疗剂。Pyk 2的小分子抑制剂在胶质母细胞瘤、其他侵袭性癌症、炎症和骨质疏松症中具有治疗应用。AlphaScreen试验将作为鉴定FERM结构域寡聚化的小分子抑制剂的主要试验。该试验之后将进行基于细胞的试验，检测全长Pyk 2活化的细胞内抑制效力。将使用基于细胞的FERM二聚化和生物化学Pyk 2催化活性测定对两种测定中确认的命中进行优先排序。将使用干粉化合物验证在寡聚化测定中显示功效但在激酶测定中无活性的命中物，并继续在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到位，我们预计快速评估命中的适用性作为起点，命中到铅研究和未来的体内评价在临床前动物模型。

 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.