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: 8084218
• 负责人: DONALD W LANDRY
• 金额: $ 49.98万
• 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/8084218
• 关键词: Adverse effects; Behavioral; Catalysis; Cocaine; Cocaine Dependence; Computational Technique; Coupled; Dose; Drug Addiction; Enzymes; Evaluation; Free Energy; Health; Hybrids; Hydrolysis; In Vitro; Investigation; Lead; Mechanics; Metabolism; Modeling; Molecular; Molecular Models; Performance; Pharmaceutical Preparations; Preclinical Testing; Process; Protocols documentation; Reaction; Rodent Model; Roentgen Rays; Site-Directed Mutagenesis; Structure; Testing; Toxic effect; 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; protein purification; 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（由申请人提供）：通过给药可卡因酯酶增强可卡因代谢已被认为是可卡因过量和成瘾的有前途的治疗策略。酯酶COCE是用于代谢天然发生的最有效的天然酶（ - ）可卡因尚未鉴定出来。通过（ - ） - 可卡因水解的催化，COCE可以预防和反向极端（ - ） - 可卡因毒性中的可卡因毒性，并且它有可能发展为（ - ） - 可卡因的毒性和行为效应的化学有用的拮抗剂。为了优化这种潜在的抗可卡因药物的功效，并最大程度地减少其可能的副作用（尤其是免疫原性），我们建议提高COCE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，剂量降低可以降低总体免疫学反应。因此，我们将重点关注COCE突变体的合理设计，发现和临床前测试，其催化效率提高了（ - ） - 可卡因。 COCE对（ - ） - 可卡因的高活动性突变体的合理设计需要对可卡因的CoCE催化水解的机制有详细的理解。可以通过使用分子建模，仿真和计算的最新计算技术进行计算研究来理解这种机制。具体目的包括：（1）通过执行量子力学（QM）计算，阐明可卡因的详细机制和反应坐标以及相应的自由能谱，用于可卡因的水解，杂种量子机械/分子/分子机械/mm/mm/mm/mm/mm/mm/mm）的杂种量子和分子验证（MOD）和分子动力学（MD）的模拟（MD）的象征（MD）（MD）的设计（MD）（MD）（2）设计，通过使用最近开发的新型计算设计方法基于过渡状态建模和模拟来计算评估大量假设的COCE突变体，然后进行湿的实验测试，包括位置定向的诱变，蛋白质表达和纯化，以及体外和体内活性测试，包括湿的实验测试。这项研究的长期目标将是最终使用COCE的高活动性突变体开发有效的抗可卡因药物。公共卫生相关性：通过给药可卡因酯酶（COCE）增强可卡因代谢，已被认为是可卡因过量和成瘾的有前途的治疗策略。在该项目中设计和发现的COCE的高活动性突变体最终将导致有效的抗可卡因药物。