HOW DIDEMNINS INHIBIT PROTEIN BIOSYNTHESIS

地德米宁如何抑制蛋白质生物合成

• 批准号: 2102889
• 负责人: PETER L. TOOGOOD
• 金额: $ 9.48万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-17 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2102889
• 关键词: Tunicata; animal extract; antineoplastics; antiviral agents; cell free system; chemical structure function; crosslink; cyclic peptides; drug design /synthesis /production; growth inhibitors; immunomodulators; inhibitor /antagonist; protein biosynthesis; radiotracer

The marine natural product didemnin B displays potent antiviral, antitumor and immunomodulatory effects in cultured cells and in vivo. It has entered phase-II clinical trails as an antitumor agent. The reasons why didemnin B displays such potent biological activity are not understood, however didemnin B has been shown to inhibit the biosynthesis of DNA, RNA and proteins in whole cells. We have demonstrated the inhibition of in vitro protein synthesis by didemnin B and didemnin A, using a Cell-free translation system derived from rabbit reticulocytes, and have shown that didemnin B is equally effective as an inhibitor of protein synthesis in wheat germ. Furthermore, this effect is attenuated by modification of didemnin B as its bis(O-acetyl)derivatives. In our preliminary results, we present data that support he hypothesis that didemnin B inhibits protein synthesis specifically at eh elongation stage. Further studies are proposed to elucidate the detailed mechanism of inhibition of protein synthesis by didemnin B in vitro. Specifically, experiments are described that will define the precise step of elongation that is affected (amino acid delivery, peptide bond formation, or translocation), based on the effect of didemnin B upon protein synthesis partial reactions in rabbit reticulocyte or yeast lysates. The cytosolic molecular target of didemnin B will be identified using radiolabeled didemnin derivatives, or by cross-linking with a didemnin ligand. The design and synthesis of a new cross-linking agent is described. The effect of didemnin B upon regulated via phosphorylation of several translation factors and ribosomal proteins. The phosphoproteins will be labeled with 32P and resolved by tow-dimensional gel electrophoresis. To identify which structural features of didemnin B are important for its inhibition of protein synthesis, didemnin analogs will be synthesized and tested as inhibitors of in vitro protein synthesis. The structure- function profile for the didemnins that is obtained from these studies will be used in the design of new potential inhibitors of translation in ukaryotes. It is hypothesized that other potent inhibitors of protein synthesis besides the didemnins will also be effective antitumor agents, and this hypothesis will be tested by the synthesis and evaluation of didemnin mimics and limited screening of related natural products.

海洋天然产品didemnin B显示有效的抗病毒， 培养细胞和体内的抗肿瘤和免疫调节作用。 它已作为抗肿瘤剂进入了II期临床步道。 这 dodemnin b显示这种有效的生物学活动的原因不是 了解，但是已显示didemnin b可以抑制生物合成 全细胞中的DNA，RNA和蛋白质的蛋白质。 我们已经证明了 抑制didemnin b和didemnI的体外蛋白质合成， 使用衍生自兔网状细胞的无细胞翻译系统， 并表明didemnin B作为抑制剂同样有效 小麦胚芽中的蛋白质合成。 此外，这种效果被减弱 通过修改Dodemnin B作为其BIS（O-乙酰基）衍生物。 在我们的 初步结果，我们提供了支持他假设的数据 dodemnin b在EH伸长延伸时特别抑制蛋白质合成 阶段。 提出了进一步的研究以阐明 在体外抑制didemnin b对蛋白质合成的抑制。 具体来说， 描述了将定义伸长的精确步骤的实验 受影响（氨基酸递送，肽键形成或 易位），基于dodemnin b对蛋白质合成的影响 兔网状细胞或酵母裂解物的部分反应。 胞质 将使用radiolabeled识别Dodemnin B的分子靶标 Didemnin衍生物，或与Didemnin配体交联。 这 描述了新的交联剂的设计和合成。 这 dodemnin b对通过几种磷酸化调节的影响 翻译因子和核糖体蛋白。 磷蛋白将是 用32p标记，并通过拖曳维凝胶电泳解决。 确定didemnin b的结构特征对其 抑制蛋白质合成，dodemnin类似物将被合成，并 被测试为体外蛋白质合成的抑制剂。 结构 - 从这些研究中获得的didemnins的功能曲线 将用于设计新的潜在翻译抑制剂 Ukaryotes。 假设其他有效的蛋白质抑制剂 除了didemnins外，合成也将是有效的抗肿瘤剂， 该假设将通过合成和评估来检验 didemnin模仿相关天然产品的有限筛选。