Novel ODC Inhibitor Phaseolotoxin in Neuroblastoma

摘要 神经母细胞瘤(NB)是一种侵袭性的儿童癌症，其中MYC同源物MYCN起着 癌基因通常被激活以驱动肿瘤的进展。NB是一种致命的肿瘤，关系到儿童的存活率 患有高危(IV期)的疾病是令人沮丧的，尽管紧张的治疗计划包括过多的高风险- 剂量化疗、外科手术、干细胞移植、放射治疗和抗体治疗。MYCN和鸟氨酸 脱羧酶(ODC)在NB中经常被解除调控，依赖于ODC的多胺控制p27/Rb- 调节肿瘤进展。MYC(N)是ODC的直接激活剂，ODC是NB的有效药物靶点 和其他MYC(N)驱动的肿瘤。二氟甲基鸟氨酸(DFMO)是目前临床上唯一的ODC抑制剂。 DFMO是FDA批准的一种抗原虫药物(锥虫病)，有效地化学预防 结肠癌，这已经进入了针对复发NB儿童的临床研究。不幸的是，DFMO是 由于其半衰期短、溶解度高、肾脏消除/清除快，因此需要非常高的剂量。 因此，寻找一种效力更高、药理特征更优越的新的ODC抑制剂是一项重要的工作 这是正当的。我们先前已经发现，天然产物相毒素(PT)抑制ODC和 在癌细胞中表现出抗增殖活性。这一提议的中心假设是，新的PT- 为本研究设计的受启发的PSorn类似物比PT或DFMO更有效的ODC抑制剂， 因此，在体内更有效地抑制了ODC依赖的NB肿瘤的生长。这项建议的目的是 确定具有最佳PK特征的最活跃的PSorn-类似物，并证实其作为一种新的 针对NB的治疗剂。在目标1中，我们将化学合成一系列PSorn类似物，用于生物和 临床前研究。我们将准备17-20个(线性和循环)PSorn类似物。在目标2中，我们将测试 假设这些新的PSorn类似物在体外是有效的ODC抑制剂。在目标3中，我们将测试疗效 PSorn类似物在基于细胞的肿瘤学模型和肿瘤异种移植研究中的应用。将选择激活的类比 通过测试漏斗，包括：ODC活性和酶动力学分析，基于NB细胞的肿瘤学 使用24个特征良好的(MYCN扩增和MYCN非扩增)Nb细胞系的模型 DFMO-小鼠不同ODC表达水平、体外ADME及移植瘤疗效的研究 耐药的NB细胞。等温量热(ITC)和X射线结晶将是最活跃的 PSorn-类似物，用于验证ODC结合结构域，并帮助进一步基于SAR的模拟优化。总而言之， 这些研究将为进一步开发一种全新的 ODC靶向治疗剂及其在治疗和预防(DFMO耐药)NB中的应用， 结肠癌和其他由MYC(N)驱动的癌症类型。

ABSTRACT Neuroblastoma (NB) is an aggressive childhood cancer in which the MYC homologue MYCN acts as an oncogene typically activated to drive tumor progression. NB is a lethal tumor and the survival rate of children with high-risk (stage IV) disease is dismal despite an intense treatment plan that includes a plethora of high- dose chemotherapies, surgery, stem cell transplant, radiation, and antibody therapy. MYCN and ornithine decarboxylase (ODC) are frequently deregulated in NB, and ODC-dependent polyamines control p27/Rb- regulated tumor progression. MYC(N) is a direct activator of ODC and ODC is a validated drug target in NB and other MYC(N)-driven tumors. Difluoromethylornithine (DFMO) is the only ODC inhibitor in the clinic today. DFMO is a FDA-approved anti-protozoan drug (trypanosomiasis) that is effective in the chemoprevention of colon cancer and that has entered clinical studies for children with relapsed NB. Unfortunately, DFMO is required at very high doses due to its short half-life, high solubility, and fast renal elimination/clearance. Therefore, the search for a new ODC inhibitor with higher potency and superior pharmacological profile is warranted. We have previously discovered that the natural product phaseolotoxin (PT) inhibits ODC and exhibits antiproliferative activity in cancer cells. The central hypothesis of this proposal is that novel PT- inspired PSorn-analogs designed for this study are more potent ODC inhibitors than PT or DFMO and, therefore, more effectively inhibit ODC-dependent NB tumor growth in vivo. The objective of this proposal is to identify the most active PSorn-analog with best PK characteristics and confirm its validity as a novel therapeutic agent against NB. In Aim 1 we will chemically synthesize a series of PSorn-analogs for biological & preclinical investigation. We will prepare 17-20 (linear & cyclic) PSorn-analogs. In Aim 2 we will test the hypothesis that these novel PSorn-analogs are potent ODC inhibitors in vitro. In Aim 3 we will test the efficacy of PSorn-analogs in cell-based oncology models and tumor xenograft studies. Active analogs will be selected through a testing funnel that includes: ODC activity and enzyme kinetic assays, NB cell-based oncology models using a panel of 24 well-characterized (MYCN amplified and MYCN non-amplified) NB cell lines with various ODC expression levels, in vitro ADME, and NB tumor xenograft efficacy studies in mice with DFMO- resistant NB cells. Isothermal calorimetry (ITC) and X-ray crystallization will be employed with most active PSorn-analogs to verify ODC binding domains and to aid further SAR-based analog optimization. In summary, these studies will provide the solid groundwork for further preclinical development of an entirely new class of ODC-targeted therapeutic agents with applications in the treatment and prevention of (DFMO-resistant) NB, colon cancer, and other MYC(N)-driven cancer types.