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: 6543505
• 负责人: Vern L. Schramm
• 金额: $ 37.76万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6543505
• 关键词: AMP deaminase; X ray crystallography; active sites; catalyst; chemical structure function; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; enzyme structure; nuclear magnetic resonance spectroscopy; nucleoside analog; ribosomal RNA; ricin; site directed mutagenesis; toxin metabolism

DESCRIPTION (provided by applicant): Recent advances in the application of kinetic isotope effects makes it possible to understand transition states for the complex interactions of RNA processing enzymes. Ricin-AB is a heterodimeric plant toxin, abundant in the castor bean. Subunit B is a galactose-specific lectin that provides cell entry to the complex followed by release of the A-chain, an adenine N-ribohydrolase specific for a single site on 28S rRNA. A single molecule of ricin is lethal for a mammalian cell, and a few ug are lethal for a human, making it among the most powerful cytotoxins. The adenine substrate resides in a hairpin stem-loop region of RNA, terminating in a GAGA tetraloop. The reaction mechanism for ricin A-chain forms a fully dissociated and transient ribooxacarbenium ion with closely related transition states. This knowledge has been used to produce the first generation of transition state analogue inhibitors for ricin A-chain, and is the most powerful catalytic site inhibitors known for the toxin. Second-generation transition state analogues for ricin a-chain will be designed from knowledge of the transition state structure. Investigation of the chemical mechanism of catalysis will use substrate specificity and site-directed mutagenesis studies. Structural analysis of ricin A-chain in complex with substrate, transition state and product analogues of RNA is intended to provide information on reaction coordinate motion and the role of individual amino acids in stabilizing the transition state complex. Powerful transition state inhibitors and chromogenic substrates for detecting ricin A-chain catalytic activity are two anticipated products of the research. These agents may be of use as rescue agents in immunochemotherapy and in detection of the toxin. The studies of ricin A-chain are intended to provide more complete knowledge of the reaction mechanisms for enzymes that recognize and covalently modify RNA. Consistent with this goal, studies will be initiated toward the transition state structure of an RNA site-specific adenylate deaminase, ADAR. Deamination at an adenylate site in mRNA produces an inosine site that is translated as a G, therefore causing A > G codon changes. Transition state inhibitors for these enzymes are anticipated to find use in altering protein expression and viral infectivity.

描述（由申请人提供）：动态同位素效应应用的最新进展使得理解RNA加工酶复杂相互作用的过渡态成为可能。蓖麻毒素ab是一种异二聚体植物毒素，在蓖麻中含量丰富。亚基B是一种半乳糖特异性凝集素，它提供细胞进入复合物，随后释放a链，这是一种针对28S rRNA上单个位点的腺嘌呤n -核糖水解酶。一个分子的蓖麻毒素对哺乳动物细胞来说是致命的，几微克对人类来说是致命的，使其成为最强大的细胞毒素之一。腺嘌呤底物位于RNA的发夹茎环区域，在GAGA四环中终止。蓖麻毒素a链的反应机制是形成一个完全解离的瞬态核碳离子，其过渡态密切相关。这一知识已被用于生产第一代蓖麻毒素a链过渡态类似抑制剂，并且是已知毒素最强大的催化位点抑制剂。蓖麻毒素a链的第二代过渡态类似物将根据过渡态结构的知识进行设计。研究催化的化学机制将使用底物特异性和定点诱变研究。对蓖麻毒素a链与底物、过渡态和RNA产物类似物的配合物进行结构分析，旨在为反应的配位运动和单个氨基酸在稳定过渡态配合物中的作用提供信息。强有力的过渡态抑制剂和显色底物检测蓖麻毒素a链催化活性是两个值得期待的研究成果。这些药物可作为免疫化疗和毒素检测的拯救剂使用。蓖麻毒素a链的研究旨在为更完整地了解识别和共价修饰RNA的酶的反应机制提供依据。与这一目标相一致，将开始研究RNA位点特异性腺苷酸脱氨酶（ADAR）的过渡态结构。mRNA中腺苷酸位点的脱胺作用产生肌苷位点，该肌苷位点被翻译为G，因此导致a > G密码子改变。这些酶的过渡态抑制剂有望用于改变蛋白质表达和病毒感染性。