PERTUSSIS, DIPHTHERIA, CHOLERA TOXINS-INHIBITOR DESIGN

百日咳、白喉、霍乱毒素抑制剂设计

• 批准号: 6497266
• 负责人: Vern L. Schramm
• 金额: $ 42.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497266
• 关键词: ADP ribosylation; G protein; chemical kinetics; chemical structure; chemical substitution; chemical synthesis; cholera toxin; diphtheria toxin; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate analog; enzyme substrate complex; pertussis toxin

This research program addresses two major and current problems in infectious disease using novel technology. Toxin-directed transition state inhibitors offer a new approach to prevent the damage of bacterial exotoxins to human tissues. Antibiotic resistance is a global problem in infectious disease. Tissue- protective toxin inhibitors could act as antibiotics which are not expected to elicit resistance in the causitive organisms. The experimental approach is to use the frontier method of enzymatic transition state analysis and to apply it to the action of bacterial exotoxins. ADP-ribosylating bacterial exotoxins catalyze the covalent modification of GTP-binding proteins. Cholera, diphtheria and pertussis toxins ADP-ribosylate Gsalpha, eukaryotic elongation factor 2, and Gialpha proteins, respectively. Transition state analysis of bacterial ADP-ribosylating exotoxins will be used to design transition state inhibitors against cholera, diphtheria, pertussis and related exotoxins. Transition-state inhibitors against bacterial exotoxins are expected to protect against the exotoxins and thus ameliorate the damage caused in these childhood and endemic diseases. Transition state structure is determined by measuring kinetic isotope effects with NAD+ substrate labeled in all of the atomic positions expected to undergo bonding changes as bonds are broken and made at the enzyme-stabilized transition state. The ADP- ribosylated G-protein is analyzed for the isotopic discrimination of the incorporated ADP-ribose. The isotope effects are then corrected to reveal the full chemical expression of intrinsic isotope effects. An atomic model of all atoms at least two bonds from the reaction center is constructed which is constrained by the values of the kinetic isotope effects. Semiemperical and ab initio methods are used to complete the structure of the transition state molecules, with constraints at every step to comply with the experimental kinetic isotope effects. Transition state structures are mapped using the molecular electrostatic potential surface at the van der Waals radius and compared to that of the substrate. The relationship provides predictive value for transition state inhibitor design. Molecules with electronic similarity close to that of the transition state are synthesized and tested as transition state inhibitors. These procedures have resulted in the discovery of novel transition state inhibitors for several simple enzymatic reactions. The goal of this work is to extend transition state inhibitor design to the complex reactions catalyzed by bacterial ADP-ribosylating toxins.

该研究计划使用新技术解决了传染病的两个主要和当前问题。 毒素指导的过渡状态抑制剂提供了一种新方法，以防止细菌外毒素对人体组织损害。 抗生素耐药性是传染病中的全球问题。 组织保护性毒素抑制剂可以充当抗生素，预计不会引起因果生物的耐药性。实验方法是使用酶促过渡态分析的前沿方法，并将其应用于细菌外毒素的作用。 ADP-核糖基化细菌外毒素催化GTP结合蛋白的共价修饰。霍乱，白喉和百日咳毒素ADP-核糖基gsalpha，真核伸长因子2和Gialpha蛋白分别。 细菌ADP-核糖基毒素的过渡状态分析将用于设计针对霍乱，白喉，百日咳和相关外毒素的过渡态抑制剂。 预期针对细菌外毒素的过渡态抑制剂有望预防外毒素，从而改善这些童年时期和地方性疾病造成的损害。过渡状态结构是通过测量在所有原子位置标记的NAD+底物的动力学同位素效应来确定的，这些原子位置预期会在键上损坏并在酶稳定的过渡状态下进行键变化。 分析了ADP核糖基化的G蛋白，以分析掺入的ADP-核糖的同位素歧视。 然后对同位素效应进行校正，以揭示固有同位素效应的完整化学表达。 所有原子的原子模型至少来自反应中心的两个键，该模型受动力学同位素效应的值的约束。 半特质和AB的初始方法用于完成过渡状态分子的结构，并在每个步骤中都具有限制，以符合实验动力学同位素效应。 使用分子静电势表面在范德华半径处映射过渡状态结构，并与基板的分子结构进行了映射。 该关系为过渡状态抑制剂设计提供了预测价值。 具有接近过渡态的电子相似性的分子被合成并测试为过渡态抑制剂。 这些程序导致发现了几种简单酶促反应的新型过渡状态抑制剂。 这项工作的目的是将过渡态抑制剂设计扩展到由细菌ADP-核糖基毒素催化的复杂反应。