Development of Novel Inducers of Non-Apoptotic Cell Death to Target Glioblastoma

描述(申请人提供)：胶质母细胞瘤(GBM)是成人最常见的原发脑肿瘤。标准治疗包括手术，然后是放射和DNA烷化剂，如替莫唑胺。然而，肿瘤几乎总是复发，中位生存期保持在1-2年之间。目前的治疗方法依赖于通过引起DNA损伤来触发细胞自杀(凋亡)，但GBM细胞的基因变化使其对凋亡刺激相对不敏感。在前一个项目期间完成的研究导致了一种独特的细胞死亡形式的鉴定，这种形式被称为甲硫氨酸，这在机械上是不同的 来自于细胞凋亡。它包括刺激巨噬细胞吞噬(细胞饮酒)以及内吞液泡运输的变化，导致大量细胞空泡化和膜完整性丧失。新的化合物被发现在广泛的GBM细胞中诱导甲硫磷代谢，包括那些耐药的替莫唑胺细胞。结构-活性研究提供了首字母缩写MOMIPP所指的先导化合物。继续这一项目的中心假设是，通过确定MOMIPP的分子靶点和开发可用于将化合物定向到GBM的纳米粒(NP)传递载体和/或靶向前药，可以实现一种新型的针对GBM的局部治疗。为了验证这一假说，本文提出了三个具体的目标：目的：1)确定MOMIPP的相关蛋白质靶点(S)。这将涉及几种互补的方法，包括使用放射性标记的MOMIPP和非活性类似物对蛋白质阵列进行差异显示分析，以及使用MOMIPP光亲和探针与质谱仪相结合来识别完整的GBM细胞中的药物结合蛋白。目的-2)开发优化MOMIPP向GBM细胞输送的策略。基本的前提是，如果MOMIPP以缓释NP制剂在当地提供，作为GBM的辅助治疗可能是最有效的。为了最大限度地减少对正常细胞的潜在毒性，将对选择性地将化合物输送到GBM的创新策略进行评估，方法是用GBM定位的多肽修饰NP或将含有可移除的GBM靶向多肽的MOMIPP前药加载到NP中。目的：评价MOMIPP在GBM异种移植模型中的毒性、药代动力学及抗肿瘤作用。最有可能选择性地将MOMIPP输送到GBM的靶向NP和前药配方将在来自wt和替莫唑胺耐药GBM细胞株或从原发人类GBM浓缩的干细胞的原位异种移植中进行测试。这些研究有望确定将抑制肿瘤进展的配方，将全身毒性或对正常神经细胞的不良影响降至最低。影响：通过验证一类新的药物可以通过一种新的非凋亡机制杀死GBM细胞，这一结果可能对GBM治疗产生实质性影响。此外，肿瘤归巢NP或可靶向于GBM的前药的开发将代表着一项技术进步，可能更广泛地应用于其他治疗剂的输送。

DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor in adults. Standard treatment involves surgery, followed by radiation and DNA alkylating agents like temozolomide. However, the tumors almost always recur, with median survival remaining in the range of 1-2 years. Current therapies depend on triggering cell suicide (apoptosis) by causing DNA damage, but genetic alterations in GBM cells make them relatively insensitive to apoptotic stimuli. Studies completed during the preceding project period led to the identification of a unique form of cell death termed 'methuosis', which is mechanistically distinct from apoptosis. It involves stimulation of macropinocytosis (cell drinking) together with changes in trafficking of endocytic vesicles, leading to massive cellular vacuolization and loss of membrane integrity. New compounds were discovered to induce methuosis in a broad spectrum of GBM cells, including those that are resistant temozolomide. Structure-activity studies have provided a lead compound referred to by the acronym MOMIPP. The Central Hypothesis underlying the continuation of this project is that a new type of localized therapy for GBM may be realized through the identification of the molecular targets of MOMIPP and the development of nanoparticle (NP) delivery vehicles and/or targeted prodrugs that can be used to direct the compound specifically to GBM. To test this hypothesis three Specific Aims are proposed: Aim-1) Identify the relevant protein target(s) of MOMIPP. This will involve several complementary approaches, including the use of radiolabeled MOMIPP and inactive analogs for differential display analysis of protein arrays, and the use of MOMIPP photoaffinity probes combined with mass spectrometry to identify drug-binding proteins in intact GBM cells. Aim-2) Develop strategies to optimize delivery of MOMIPP to GBM cells. The underlying premise is that MOMIPP might be most effective as an adjuvant therapy for GBM if delivered locally in a sustained release NP formulation. To minimize potential toxicity to normal cells, innovative strategies will be evaluated for selective delivery of the compound to GBM by decorating the NP with GBM-homing peptides or loading them with a MOMIPP prodrug containing a removable GBM-targeting peptide. Aim-3) Evaluate the toxicity, pharmacokinetic properties and anti-tumor efficacy of MOMIPP in GBM xenograft models. Targeted NP and prodrug formulations with the greatest potential for selective delivery of MOMIPP to GBM will be tested in orthotopic xenografts derived from wt and temozolomide-resistant GBM cell lines or stem cells enriched from primary human GBM. These studies are expected to identify formulations that will inhibit tumor progression with minimal systemic toxicity or ill effects on normal neural cells. Impact: The results could have a substantial impact on GBM therapy by validating a new class of drugs that can kill GBM cells by a novel non-apoptotic mechanism. In addition, the development of tumor-homing NP or prodrugs that can be targeted to GBM would represent a technological advance that might be applied more generally for delivery of other therapeutic agents.