STRUCTURE AND FUNCTION OF HUMAN ANGIOGENIN

人血管生成素的结构和功能

• 批准号: 2229258
• 负责人: ROBERT SHAPIRO
• 金额: $ 23.81万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2229258
• 关键词: X ray crystallography; active sites; adenosine diphosphate; angiogenesis; angiogenesis factor; cell adhesion; chemical kinetics; chemical synthesis; conformation; cytosine nucleotides; enzyme activity; enzyme complex; enzyme mechanism; enzyme substrate; esterase inhibitor; fluorescence spectrometry; growth inhibitors; mutant; nucleic acid inhibitor; pancreatic ribonuclease; protein sequence; receptor binding; site directed mutagenesis; tissue /cell culture

Human angiogenin (Ang) is an unusual member of the pancreatic ribonuclease (RNase) superfamily that induces blood vessel formation in vivo. Recent studies have demonstrated that Ang plays a critical role in the establishment and growth of some human tumors. These results define as a medically important goal the identification or design of Ang inhibitors suitable for clinical applications. The approach to this problem presented here is based on recent breakthroughs in determining the 3D structure of Ang, achieved together with our collaborators, and on the continuing availability of new crystallographic data. Three classes of Ang antagonists will be developed: (1) Small nucleotide-based inhibitors of the ribonucleolytic activity of Ang that is known to be essential for its angiogenic action. The design of these compounds will take into account the unexpected blockage of the pyrimidine binding site observed in the Ang crystal structure (and confirmed by mutagenesis), and the consequent requirement for Ang to undergo an extensive conformational change in order to act on RNA. The nature of this conformational change will be explored by X-ray crystallography of Ang-nucleotide complexes, fluorescence spectroscopy, and mutagenesis. New nucleotide inhibitors will be synthesized and tested based on these findings and on kinetic results obtained with RNase A inhibitors and related molecules. These efforts will be aided by the mutagenic characterization of inhibitor/substrate binding subsites on Ang and by the development of an improved enzymatic assay for Ang. The antiangiogenic and antitumor activities of any inhibitors with low I/i values will be examined. The possibility will be investigated that molecules other than RNA are substrates for Ang; if so, additional inhibitors will be designed based on these substrates. (2) Derivatives of placental RNase inhibitor (PRI), an extremely tight- binding protein inhibitor of the enzymatic and angiogenic activities of Ang. Contact residues on PRI will be identified by X-ray crystallography and mutagenesis. The inhibitory properties of PRI domains containing critical residues will be tested. Mutagenesis will be used to improve the specificity and stability of PRI or any smaller active derivatives obtained. (3) Inhibitors of cellular interactions of Ang that are also required for angiogenic activity. Residues and regions of Ang that interact with Ang- binding proteins/receptors on endothelial cells will be identified by testing the capacities of Ang mutants to bind to these sites. The results will help to establish the mutual relationships of the enzymatic, angiogenic, and various cell-binding properties of Ang. The 3D structures of the complexes of Ang with any cell-surface Ang-binding proteins that can be obtained in quantity will be examined. The capacity of peptides derived from Ang/Ang-binding protein contact regions to inhibit the angiogenic activity of Ang will be tested.

人血管生成素 (Ang) 是胰腺核糖核酸酶的一个不寻常成员 （RNase）超家族，诱导体内血管形成。 最近的 研究表明，Ang 在 一些人类肿瘤的建立和生长。 这些结果定义为 医学上重要的目标是血管紧张素抑制剂的识别或设计 适合临床应用。 解决这个问题的方法 这里介绍的是基于最近在确定 3D Ang 的结构，与我们的合作者一起实现，并且关于 新晶体学数据的持续可用性。 三类 将开发血管紧张素拮抗剂： (1) 核糖核酸酶活性的基于小核苷酸的抑制剂 众所周知，Ang对其血管生成作用至关重要。 设计 这些化合物将考虑到意外堵塞 在 Ang 晶体结构中观察到的嘧啶结合位点（和 通过诱变证实），以及随之而来的要求 Ang 经历广泛的构象变化以作用于RNA。 这 这种构象变化的本质将通过 X 射线来探索 Ang-核苷酸复合物的晶体学、荧光光谱、 和诱变。 新的核苷酸抑制剂将被合成和测试 基于这些发现和 RNase A 获得的动力学结果 抑制剂及相关分子。 这些努力将得到以下机构的协助 Ang 上抑制剂/底物结合亚位点的诱变特征 并开发了一种改进的 Ang 酶测定法。 这 任何低 I/i 抑制剂的抗血管生成和抗肿瘤活性 值将被检查。 将调查这种可能性 RNA以外的分子是Ang的底物；如果是这样，则额外 抑制剂将根据这些底物进行设计。 (2) 胎盘核糖核酸酶抑制剂 (PRI) 的衍生物，一种极其紧密的 酶促和血管生成活性的结合蛋白抑制剂 安。 PRI 上的接触残留物将通过 X 射线晶体学进行鉴定 和诱变。 PRI 结构域的抑制特性包含 将测试关键残留物。 诱变将用于改善 PRI 或任何较小的活性衍生物的特异性和稳定性 获得。 (3) Ang 细胞相互作用的抑制剂，这也是 血管生成活性。 与 Ang-相互作用的 Ang 残基和区域 内皮细胞上的结合蛋白/受体将通过以下方式鉴定 测试 Ang 突变体结合这些位点的能力。 结果 将有助于建立酶的相互关系， 血管生成和 Ang 的各种细胞结合特性。 3D 结构 Ang 与任何细胞表面 Ang 结合蛋白的复合物 将检查是否可以大量获得。 肽的容量 源自 Ang/Ang 结合蛋白接触区域，以抑制 将测试Ang的血管生成活性。