Novel small-molecule inhibitors of Wee1 kinase for medulloblastoma treatment

描述（申请人提供）：髓母细胞瘤是儿童最常见的原发性脑肿瘤。目前髓母细胞瘤的治疗包括手术切除、放疗和DNA烷基化剂（如顺铂）化疗。虽然这种治疗方法提高了生存率，但需要高剂量的细胞毒性化疗来规避耐药机制并赋予临床疗效，不幸的是，这通常导致持久的神经认知缺陷，发育迟缓，耳聋，甚至继发性肿瘤。必须提高化疗的特异性和疗效，以安全地减少剂量和减少药物相关的不良反应。此外，虽然目前对标准风险患者的治疗已经改善了生存率，但仍有一部分Myc扩增的高危患者预后极差，5年生存率低于30%。因此，更深入地了解成神经管细胞瘤的分子通路对于开发更特异性的新药以改善高危患者的预后至关重要。通过对成神经管细胞瘤细胞进行基因表达通路分析和全基因组siRNA筛选，我们确定了Wee1激酶作为成神经管细胞瘤潜在的新分子靶点。Wee1激酶参与G2-M检查点，在DNA损伤的情况下防止有丝分裂，因此可能在对DNA烷基化剂的耐药性中发挥作用。我们的初步数据表明，Wee1可以防止顺铂引起的DNA损伤引起的细胞死亡，并且已知的Wee1抑制剂MK1775与顺铂具有协同活性。然而，MK1775的选择性尚未得到严格的评估，它似乎与血浆蛋白结合的潜力很高，并且通过血脑屏障扩散的能力降低，限制了它在临床治疗脑癌的有效性。因此，本研究的中心目标是确定小分子与Wee1结合的结构要求，以开发具有改进药物性能的新型Wee1选择性抑制剂，并研究在成神经管细胞瘤中靶向Wee1的潜力。我们建议通过基于计算的对接和体外活性和结合试验筛选194个激酶抑制剂的SelleckChem库。这些筛选的数据将相互关联，以确定活性化合物的子集，这些活性化合物将用作抑制剂设计的基础。Wee1缺乏HRD和DFG基序的事实为选择性靶向提供了机会。我们将合成一系列新的抑制剂，并在我们的细胞系统中与MK1775并行测试它们。我们将评估这些抑制剂作为单一药物对肿瘤细胞生长的影响，并测试这些抑制剂是否与顺铂显示协同活性。此外，我们将确定p53是否在髓母细胞瘤中介导对Wee1抑制的敏感性，以及Wee1抑制是否在具有高Myc扩增的肿瘤中具有治疗潜力，这是高风险患者的特征。然后，我们将把我们的研究扩展到体内模型，以确定Wee1抑制剂的药代动力学和组织分布，以及它们对异种移植物模型中肿瘤生长的影响。

DESCRIPTION (provided by applicant): Medulloblastoma is the most common primary brain tumor in children. Current treatment for medulloblastoma includes surgical resection, radiation and chemotherapy with DNA alkylating agents, such as cisplatin. Although this approach to therapy has improved survival rates, high doses of cytotoxic chemotherapy are required to circumvent drug resistance mechanisms and to confer clinical efficacy, and unfortunately this often results in lasting neurocognitive defects, stunted growth, deafness, and even secondary tumors. The specificity and efficacy of chemotherapy must be improved to safely allow dose reductions and reduce drug-related adverse effects. Furthermore, while current therapy for standard-risk patients has resulted in improved survival rates, there is a subset of high-risk patients with Myc amplification who continue to have an extremely poor prognosis with a 5-year survival rate of less than 30%. Therefore, a more thorough understanding of the molecular pathways in medulloblastoma is critical for the development of more specific novel drugs to improve outcomes in high-risk patients. We identified Wee1 kinase as a potential new molecular target for medulloblastoma from an integrated genomic analysis using pathway analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells. The Wee1 kinase participates in the G2-M checkpoint to prevent mitosis in the presence of DNA damage and therefore may play a role in drug resistance to DNA alkylating agents. Our preliminary data indicate that Wee1 prevents DNA damage-induced cell death by cisplatin and that the known Wee1 inhibitor MK1775 displays synergistic activity with cisplatin. However, the selectivity of MK1775 has not been rigorously assessed and it appears to have a high potential for binding to plasma protein and has a reduced capacity to diffuse across the blood brain barrier, restricting its usefulness in the clinic for the treatment of brain cancers. Therefore, a central aim of this research is to determine the structural requirements for small molecule binding to Wee1 to develop novel Wee1 selective inhibitors with improved drug properties and examine the potential of targeting Wee1 in medulloblastoma. We propose to screen the SelleckChem library of 194 kinase inhibitors by computational-based docking and in in vitro activity and binding assays. The data from these screens will be correlated to identify a subset of active compounds which will be used as a basis for inhibitor design. The fact that Wee1 lacks both the HRD and DFG motifs presents the opportunity for selective targeting. We will synthesize a small series of novel inhibitors and test them in parallel with MK1775 in our cell-based systems. We will evaluate the effect of these inhibitors on tumor cell growth as single agents and test if these inhibitors display synergistic activity with cisplatin. In addition, we will determine if p53 playsa role in mediating sensitivity to Wee1 inhibition in medulloblastoma and if Wee1 inhibition has therapeutic potential in tumors that have high Myc amplification which is characteristic of high risk patients. We will then extend our studies into in vivo models to determine the pharmacokinetics and tissue distribution of the Wee1 inhibitors and their effect on tumor growth in our xenograft model.