BINDING OF ANTIPROLIFERATIVE N ALKYL POLYAMINES TO TRNA: AN NMR STUDY

抗增殖 N 烷基多胺与 TRNA 的结合：核磁共振研究

• 批准号: 6281556
• 负责人: Venudhar K Reddy
• 金额: $ 0.65万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281556
• 关键词: biological products; biomaterials; biomedical resource; neoplasm /cancer; nuclear magnetic resonance spectroscopy; nucleic acids; technology /technique

The naturally occurring polyamines, spermidine and spermine, have been shown to be critical for cell growth and carcinogenesis. Their N-bisethyl synthetic analogs and homologs were found to be strongly inhibitory of cell proliferation in many human tumor cell lines, some of which were chemoresistant to other therapeutic agents. Even though the cell killing effects of several of the bisethyl derivatives is so efficient that some of them have reached the stage of clinical trials, the molecular rationale behind these effects is still unknown. This proposal focuses on the clarification of this problem. It is known that polyamines govern the extent and fidelity of the translation of the genetic code by binding to tRNA, but the ways in which this interaction proceeds are still unexplained. We have recently shown, using 15N NMR, that the natural polyamines and the synthetic N-bisethyl analogs with antiproliferative effects bind to tRNA through hydrogen bonds; to elucidate how the nature of these interactions and to pinpoint the domains of tRNA which bind to the polyamines we propose to use additional novel NMR techniques. This will help fill a basic scientific gap arising from the fact that currently available X-ray and solution NMR data afford conflicting results concerning these systems. Our analyses on polyamine/tRNA will use 2D NOSEY and ROESY solution NMR techniques to locate the sites of binding as a function of increasing relative concentrations of polyamines; these studies will involve yeast and E. coli tRNAPhe, as well as thirteen natural and therapeutically-related polyamines. The 9.5-15 ppm region of the 1H NMR spectrum (imino protons) of E. coli and yeast tRNAPhe will be used to monitor the sites of binding, by measuring Overhauser cross peaks between the 1H imino resonances from the macromolecule and the polyamine protons. Since all the 1H NMR studies that have been so far carried out on tRNA revealed significant changes in the spectral parameters of at least some imino protons upon addition of polyamines, our spectroscopic approach will provide highly reliable information about the location of the binding sites. The Overhauser experiments will use polyamines enriched with 13C in the carbons bonded to the amino groups. This will allow us to acquire 13C-half-filtered 2D NMR spectra where only protons bound to 13C heteroatoms appear along one of the frequency dimensions. We expect to characterize from these measurements a binding interaction that reflects the X-ray diffraction results as low polyamine/tRNA rations, while explaining the NMR behaviour of base-paired imino protons as relative polyamine concentrations increase. If our hypotheses are confirmed, the NMR analyses will detect the interaction of therapeutically-relevant polyamines with specific sites of the tRNA backbone. These results will be a decisive contribution toward the development of new synthetic polyamines to control cell protein synthesis.

天然存在的多胺、亚精胺和精胺，具有 已被证明对细胞生长和致癌作用至关重要。 他们的 N-二乙基合成类似物和同系物被发现具有很强的 抑制许多人类肿瘤细胞系的细胞增殖，某些 其中对其他治疗剂具有化学抗性。 虽然 几种双乙基衍生物的细胞杀伤作用是如此 高效，其中一些已进入临床试验阶段， 这些效应背后的分子原理仍然未知。 这 提案的重点是澄清这个问题。 我们都知道 多胺控制着翻译的程度和保真度 通过与 tRNA 结合来编码遗传密码，但是这种方式 互动收益仍然无法解释。 我们最近展示了， 15N NMR 表明天然多胺和合成多胺 具有抗增殖作用的 N-二乙基类似物通过与 tRNA 结合 氢键；阐明这些相互作用的性质和 为了查明与多胺结合的 tRNA 结构域，我们 建议使用其他新颖的核磁共振技术。 这将有助于填补 由于目前可用的技术而产生的基本科学差距 X 射线和溶液 NMR 数据提供了相互矛盾的结果 这些系统。 我们对多胺/tRNA 的分析将使用 2D NOSY 和 ROESY 解决方案 NMR 技术可定位结合位点 增加多胺相对浓度的函数；这些 研究将涉及酵母和大肠杆菌 tRNAPhe，以及 13 种 天然和治疗相关的多胺。 9.5-15 ppm 区域 大肠杆菌和酵母 tRNAPhe 的 1H NMR 谱（亚氨基质子） 将用于通过测量 Overhauser 来监测结合位点 来自大分子的 1H 亚氨基共振之间的交叉峰 多胺质子。 由于所有 1H NMR 研究都如此 对 tRNA 进行的进一步研究揭示了光谱的显着变化 添加多胺时至少一些亚氨基质子的参数， 我们的光谱方法将提供高度可靠的信息 关于结合位点的位置。 奥奥豪泽实验 将使用与键合的碳中富含 13C 的多胺 氨基。 这将使我们能够获得 13C 半过滤 2D NMR 仅与 13C 杂原子结合的质子出现在光谱中 的频率维度。 我们期望从这些特征中 测量反映 X 射线衍射的结合相互作用 结果为低多胺/tRNA 比率，同时解释了 NMR 碱基配对亚氨基质子作为相对多胺的行为 浓度增加。 如果我们的假设得到证实，核磁共振 分析将检测治疗相关的相互作用 具有 tRNA 主链特定位点的多胺。 这些结果 将为新时代的发展做出决定性的贡献 合成多胺来控制细胞蛋白质的合成。