Novel ODC Inhibitor Phaseolotoxin in Neuroblastoma

摘要 神经母细胞瘤（NB）是一种侵袭性儿童癌症，其中MYC同源物MYCN作为一种免疫抑制剂。 癌基因通常被激活以驱动肿瘤进展。NB是一种致死性肿瘤， 高风险（第四阶段）疾病是令人沮丧的，尽管密集的治疗计划，包括过多的高- 剂量化疗、手术、干细胞移植、放射和抗体治疗。MYCN和鸟氨酸 脱羧酶（ODC）在NB中经常失调，而依赖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中经验证的药物靶点 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批准的抗原生动物药物（锥虫病），可有效地化学预防 结肠癌，并已进入临床研究的儿童复发性NB。不幸的是，DFMO 由于其半衰期短、溶解度高和快速肾消除/清除，需要极高剂量。 因此，寻找具有更高效力和上级药理学特征的新的ODC抑制剂是非常重要的。 有正当理由我们以前已经发现，天然产物菜豆毒素（PT）抑制ODC， 在癌细胞中表现出抗增殖活性。这一建议的核心假设是，新的PT- 为该研究设计的受启发的PSorn类似物是比PT或DFMO更有效的ODC抑制剂， 因此，在体内更有效地抑制依赖ODC的NB肿瘤生长。这项建议的目的是 鉴定具有最佳PK特征的最具活性的PSorn类似物，并确认其作为新的 抗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。我们将制备17-20（线性和环状）PSorn-类似物。在目标2中，我们将测试 假设这些新的PSorn类似物是体外有效的ODC抑制剂。在目标3中，我们将测试 PSorn类似物在基于细胞的肿瘤模型和肿瘤异种移植研究中的应用。将选择活性类似物 通过测试漏斗，包括：ODC活性和酶动力学测定，NB细胞肿瘤学 使用一组24个充分表征的（MYCN扩增和MYCN非扩增）NB细胞系的模型， 在DFMO小鼠中进行的各种ODC表达水平、体外ADME和NB肿瘤异种移植物功效研究。 NB细胞。等温量热法（ITC）和X-射线结晶将与最活跃的 PSorn-类似物，以验证ODC结合结构域并帮助进一步基于SAR的类似物优化。总的来说， 这些研究将为进一步临床前开发一类全新的 具有在治疗和预防（DFMO-抗性）NB中的应用的ODC-靶向治疗剂， 结肠癌和其他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.