High-activity mutants of cocaine esterase for treatment of drug addiction

用于治疗药物成瘾的可卡因酯酶高活性突变体

• 批准号: 7674510
• 负责人: DONALD W LANDRY
• 金额: $ 49万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674510
• 关键词: Adverse effects; Arts; Behavioral; Catalysis; Cocaine; Cocaine Dependence; Computational Technique; Coupled; Dose; Drug Addiction; Enzymes; Evaluation; Free Energy; Hybrids; Hydrolysis; In Vitro; Investigation; Lead; Mechanics; Metabolism; Modeling; Molecular; Molecular Models; Overdose; Performance; Pharmaceutical Preparations; Preclinical Testing; Process; Protocols documentation; Reaction; Rodent Model; Roentgen Rays; Site-Directed Mutagenesis; Structure; Testing; Toxic effect; addiction; base; cocaine esterase; cocaine overdose; cocaine use; computer studies; design; esterase; immunogenicity; improved; in vivo; models and simulation; molecular dynamics; molecular modeling; mutant; novel; preclinical study; prevent; protein expression; public health relevance; quantum; response; therapeutic effectiveness; three dimensional structure; treatment strategy

DESCRIPTION (provided by applicant): Enhancing cocaine metabolism by administration of cocaine esterases has been recognized as a promising treatment strategy for cocaine overdose and addiction. The esterase CocE is the most efficient native enzyme for metabolizing naturally occurring (-)-cocaine yet identified. Through catalysis of (-)-cocaine hydrolysis, CocE can both prevent and reverse extreme (-)- cocaine toxicity in rodent models and it has the potential to be developed into a chemically useful antagonist of the toxic and behavioral effects of (-)-cocaine. In order to optimize the efficacy of this potential anti-cocaine medication and minimize its possible side effects (particularly immunogenicity), we propose to improve the catalytic efficiency of CocE against (-)- cocaine. The higher the catalytic efficiency of the enzyme against (-)-cocaine, the lower the dose required to achieve therapeutic effectiveness and the decrease in dose can reduce the overall immunological response. Hence we will focus on the rational design, discovery, and preclinical testing of CocE mutants with an improved catalytic efficiency against (-)-cocaine. The rational design of high-activity mutants of CocE against (-)-cocaine requires a detailed understanding of the mechanism for CocE-catalyzed hydrolysis of cocaine. This mechanism can be understood by performing computational studies using the state-of-the-art computational techniques of molecular modeling, simulation, and calculation. The specific aims include: (1) Elucidation of the detailed mechanism and reaction coordinate and the corresponding free energy profiles for CocE-catalyzed hydrolysis of cocaine by performing quantum mechanical (QM) calculations, hybrid quantum mechanical/molecular mechanical (QM/MM) calculations, and molecular dynamics (MD) simulations, etc. (2) Design, discovery, and testing of CocE mutants with an improved catalytic efficiency against (-)-cocaine by using a recently developed novel computational design approach based on the transition state modeling and simulation to computationally evaluate a large number of hypothetical CocE mutants, followed by wet experimental tests including site-directed mutagenesis, protein expression and purification, and in vitro and in vivo activity tests. The long-term objective of this investigation will be to eventually develop an efficient anti-cocaine medication using a high-activity mutant of CocE. PUBLIC HEALTH RELEVANCE: Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction. The high-activity mutants of CocE to be designed and discovered in this project will eventually lead to an efficient anti-cocaine medication.

描述（由申请方提供）：通过给予可卡因酯酶来增强可卡因代谢已被认为是可卡因过量和成瘾的一种有前景的治疗策略。酯酶CocE是迄今发现的用于代谢天然存在的（-）-可卡因的最有效的天然酶。通过催化（-）-可卡因水解，CocE可以预防和逆转啮齿动物模型中的极端（-）-可卡因毒性，并且有潜力开发成为（-）-可卡因毒性和行为效应的化学有用拮抗剂。为了优化这种潜在抗可卡因药物的功效并最大限度地减少其可能的副作用（特别是免疫原性），我们建议提高CocE针对（-）-可卡因的催化效率。酶对（-）-可卡因的催化效率越高，实现治疗效果所需的剂量越低，并且剂量的降低可以降低总体免疫应答。因此，我们将集中在合理的设计，发现和临床前测试的CocE突变体与改善的催化效率对（-）-可卡因。合理设计高活性的CocE突变体需要对CocE催化可卡因水解的机制有详细的了解。这种机制可以通过使用最先进的分子建模、模拟和计算的计算技术进行计算研究来理解。具体目标包括：（1）通过量子力学（QM）计算、混合量子力学/分子力学（QM/MM）计算和分子动力学（MD）模拟等，阐明CoE催化可卡因水解的详细机理和反应坐标以及相应的自由能分布。以及测试具有改进的针对（-）-可卡因通过使用最近开发的基于过渡态建模和模拟的新型计算设计方法来计算评估大量假设的CocE突变体，然后进行湿实验测试，包括定点诱变，蛋白表达和纯化，以及体外和体内活性测试。这项研究的长期目标将是最终开发一种有效的抗可卡因药物，使用高活性的CocE突变体。公共卫生关系：通过施用可卡因酯酶（CocE）来增强可卡因代谢已被认为是治疗可卡因过量和成瘾的有希望的策略。本项目中设计和发现的高活性CocE突变体将最终导致有效的抗可卡因药物。