SYNTHESIS & USE OF TRITIATED DIDEMNINS

合成

• 批准号: 6119739
• 负责人: HIROMI MORIMOTO
• 金额: $ 0.52万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6119739
• 关键词: biological products; biomedical resource

The naturally occurring cyclic peptide didemnin B displays antineoplastic activity against human tumor cell lines in culture and weak antitumor activity in human patients. Didemnin B was the first marine natural product to enter clinical trials for treating cancer. Unfortunately, it has not fulfilled its early promise in this regard, possibly due to rapid metabolism of the compound in vivo. However, didemnin B also possesses immunosuppressive activity and prolongs graft survival in rats and mice. This immunosuppressive activity occurs at concentrations of didemnin B that are significantly lower than necessary to inhibit cell growth and may be linked to the ability of this compound to halt cell cycle progression at the G1-S border. Structure-function studies of synthetic and naturally occurring didemnins have identified features of these molecules that are important for its different biological properties; these data suggest that the immunomodulatory effects and cytotoxic effects of didemnins may occur through two distinct mechanisms. TITLE: Synthesis and Use of Tritiated Didemnins (Continued) The cytotoxic properties of didemnin B have been attributed to the ability of this molecule to inhibit protein biosynthesis. Consistent with this hypothesis, we have shown that didemnin B inhibits translation in vitro by interfering with ribosomal translocation. Inhibition of translation in vitro requires a protein called elongation factor-1a (eEF-1a), implicating this translation factor as a target for didemnin B binding. In support of this hypothesis, eEF-1a was isolated from brain tissue using a didemnin affinity column, and a didemnin derivative was shown to bind to this protein in a GTP-dependent manner with Kd = 15 mM. Despite these successes, some uncertainty exists regarding the relevance of these findings to the mechanism of action of didemnin B in cells. For example, both the dissociation constant for didemnin binding to eEF-1a, and the IC50 for inhibition of protein synthesis in vitro, are three orders of magnitude higher than the concentration of didemnins required to inhibit cell growth. Moreover, some of the biological effects of didemnins are achieved in the absence of any significant effect upon total protein synthesis; notably (1) didemnin B inhibits T-cell blastogenesis at concentrations approximately three orders of magnitude lower than are required to inhibit protein biosynthesis in the same cells, and (2) vasopressin stimulated myo-inositol uptake by WRK1 (rat mammary tumor) cells is inhibited by nordidemnin at concentrations that have no significant effect upon protein synthesis. These observations may reflect the operation of even more than two mechanisms for didemnins in cells, which would be consistent with the idea that the cytotoxic and immunosuppressive effects of didemnins arise through different pathways. One or more of these pathways must account also for the recent discovery that didemnin B induces programmed cell death in both human and murine tumor cell lines. A second didemnin binding protein has been isolated using an affinity column similar to the one used to isolate eEF-1a. This protein displays protein pamitoyl thioesterase activity and has been linked to the severe brain disorder, infantile neuronal ceroid lipofuscinosis. However, the significance of this protein to the biological properties of didemnins is presently unclear. Thus, despite the clinical potential of didemnins as antitumor and immonusuppressive agents, many questions remain to be answered regarding their mechanism of action. Experiments to address these questions would be facilitated by the availability of radioactive, biologically functional didemnin analogs. The basic molecule was labelled by heterogeneous tritiation of a double bond in the dipeptide fragment, and coupling to the main part of the molecule. The product was then purified by HPLC, and analyzed by proton and tritium NMR spectroscopy.

天然发生的环状肽dodemnin b显示 在培养和 人类患者的抗肿瘤活性弱。 Didemnin B是第一个 海洋天然产品进入治疗癌症的临床试验。 不幸的是，它在这方面尚未实现其早期承诺， 可能是由于体内化合物的快速代谢。 然而， Dodemnin B还具有免疫抑制活动并延长 大鼠和小鼠的移植物存活。 这种免疫抑制活性 发生在dodemnin b的浓度下发生明显降低 抑制细胞生长所必需的，可能与能力有关 该化合物在G1-S边界停止细胞周期的进程。 合成和自然发生的结构功能研究 didemnins已经确定了这些分子的特征 对其不同的生物学特性很重要；这些数据暗示 Dodemnins的免疫调节作用和细胞毒性作用 可能通过两种不同的机制发生。 标题：合成和使用 tritived dodemnins（续）的细胞毒性特性 didemnin b已归因于该分子的能力 抑制蛋白质生物合成。 与这个假设一致，我们 已经表明didemnin b可以在体外抑制翻译 干扰核糖体易位。 抑制翻译 体外需要一种称为伸长因子1a（EEF-1A）的蛋白质， 将这一翻译因素牵连为Didemnin b的目标 结合。 为了支持这一假设，将EEF-1A从 使用dodemnin亲和力柱和dodemnin的脑组织 衍生物显示以GTP依赖性方式与该蛋白结合 KD = 15毫米。 尽管取得了这些成功，但仍然存在一些不确定性 关于这些发现与行动机理的相关性 细胞中的didemnin b。 例如，离解常数 对于didemnin与EEF-1A结合，以及抑制蛋白质的IC50 体外合成，比三个数量级高三个数量级 需要抑制细胞生长所需的dodemnins浓度。 而且， Didemnins的某些生物学作用是在 对总蛋白质合成没有任何显着影响； 值得注意的（1）diDemnin b抑制浓度下的T细胞囊泡发生 比三个数量级低约三个数量级 抑制同一细胞中蛋白质的生物合成，（2）加压素 WRK1（大鼠乳腺肿瘤）细胞刺激了肌醇的肌醇摄取 北丁宁以没有显着的浓度抑制 对蛋白质合成的影响。 这些观察结果可能反映了 在细胞中的didemins的两种以上机制的操作， 这与细胞毒性和 Dodemnins的免疫抑制作用是通过不同的 途径。 这些途径中的一个或多个也必须说明 最近发现的发现dodemnb诱导了两者的编程细胞死亡 人和鼠肿瘤细胞系。 第二个didemnin结合蛋白 已经使用与曾经的亲和柱进行隔离 分离EEF-1A。 该蛋白质显示蛋白质pamitoyl硫酯酶 活动，与严重的脑疾病，婴儿有关 神经元粘膜脂肪铁病。 但是，这一点的意义 目前，Dodemnins的生物学特性的蛋白质是 不清楚。 因此，尽管dodemnins具有临床潜力 抗肿瘤和免疫抑制剂，许多问题仍然是 回答了他们的作用机理。 实验要解决 这些问题将通过可用性来促进 放射性，生物功能性的dodemn类似物。 基本 分子通过双键在异质上进行标记 二肽片段，并耦合到分子的主要部分。 然后通过HPLC纯化产品，并通过质子和 NMR光谱法。