RICIN--MECHANISM, TRANSITION STATE AND INHIBITOR DESIGN

蓖麻毒素--机制、过渡态和抑制剂设计

• 批准号: 6172924
• 负责人: Vern L. Schramm
• 金额: $ 35.29万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6172924
• 关键词: X ray crystallography; active sites; chemical bond; chemical structure function; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; hydrolysis; molecular orbital; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; protonation; ricin; solvolysis; toxin metabolism

Ricin is an RNA-depurinating toxin found in the castor bean. A single adenine is hydrolyzed from 28S rRNA, rendering the ribosome inactive. One molecule is lethal for mammalian cells, making it one of the most toxic biological molecules. The cytotoxicity of ricin is now being exploited in clinical trials to destroy unwanted cells. Despite the novel catalytic properties and potential clinical uses of ricin, little is known of it's substrate specificity, catalytic mechanism or transition state structure. The only inhibitors with Km/Ki greater than 1 have been prepared by this laboratory. Recent advances in the application of kinetic isotope effects to enzymatic reactions has permitted the characterization of the major features of several N-ribohydrolase enzymatic transition states. Ricin A-chain catalyzes a reaction chemically similar to nucleoside and nucleotide N-ribohydrolases and is a candidate for similar analysis. Availability of transition state information proves fundamental information of the catalytic mechanism and has assisted in chemical mechanisms and transition state structure for the catalytic subunit of ricin, ricin A chain. This information will be applied to the design of molecules which are inhibitors of the enzyme. The proposed inhibitors will be synthesized and characterized by kinetic and binding experiments. Stem-loop RNA and hybrid inhibitor molecules will be synthesized to define substrate and inhibitor specificity. Selected stem- loop RNA structures will be solved by NMR. Incorporation of a spin- label at the depurination site of stem-loop RNA analogues will be used to provide a binding probe and to permit mapping of the protein-RNA geometry in the catalytic site cavity. The transition state structure will be investigated by kinetic isotope effects. Substrate RNA analogues will be synthesized to test if solvolysis of the N-ribosidic bond depends on leaving group activation (acid-catalyzed solvolysis), ribosyl activation (ribooxocarbenium ion stabilization) or ribosyl hydroxyl ionization (base-catalyzed solvolysis) or a combination of more than one of these mechanisms. Inhibitors which bind tightly will be characterized by cocrystalization with ricin A-chain for x-ray crystal studies.

蓖麻毒素是一种在蓖麻子中发现的RNA脱嘌呤毒素。 单个 腺嘌呤从28 S rRNA水解，使核糖体 不活跃。 其中一种分子对哺乳动物细胞是致命的， 毒性最强的生物分子 蓖麻毒素的细胞毒性 在临床试验中被用来破坏不需要的细胞。 尽管 蓖麻毒素的新催化特性和潜在的临床用途， 已知其底物特异性、催化机制或过渡 国家结构。 Km/Ki大于1的唯一抑制剂有 是由这个实验室准备的。 动力学同位素效应在核反应中应用的新进展 酶促反应允许表征主要的 几种N-核糖水解酶的酶促过渡状态的特征。 蓖麻毒素 A链催化的反应在化学上类似于核苷， 核苷酸N-核糖水解酶，并且是类似分析的候选者。 过渡态信息的可用性证明是至关重要的 催化机理的信息，并协助化学 催化亚基的过渡态结构 蓖麻毒素，蓖麻毒素A链。 这些信息将应用于设计 作为酶抑制剂的分子。 拟议的抑制剂 将被合成，并通过动力学和结合来表征 实验 茎环RNA和杂合抑制剂分子将被 合成以确定底物和抑制剂特异性。 选择的茎- 环RNA结构将通过NMR解析。 加入一个spin- 将使用茎环RNA类似物脱嘌呤位点的标记 以提供结合探针并允许蛋白质-RNA的作图 催化位点空腔中的几何形状。 过渡国家结构将 通过动力学同位素效应进行研究。 底物RNA类似物 将被合成，以测试N-核苷键的溶剂分解是否依赖于 在离去基团活化（酸催化的溶剂分解）、核糖基活化 （核糖氧碳正离子稳定化）或核糖基羟基电离 （碱催化溶剂分解）或这些中的一种以上的组合 机制等 紧密结合的抑制剂的特征在于： 与蓖麻毒素A链共结晶，用于X射线晶体研究。