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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

基本信息

批准号：
8286381
负责人：
DONALD W LANDRY
金额：
$ 54.92万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-15 至 2016-06-30
项目状态：
已结题

项目摘要

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)：通过给予可卡因酯酶来增强可卡因代谢已被公认为是治疗可卡因过量和成瘾的一种有前途的策略。酯酶Coce是目前已知的代谢天然(-)可卡因的最有效的天然酶。Coce通过催化(-)-可卡因的水解，可以预防和逆转(-)-可卡因在啮齿动物模型中的极端毒性，并有可能发展成为(-)-可卡因毒性和行为效应的化学上有用的拮抗剂。为了优化这种潜在的抗可卡因药物的疗效，并将其可能的副作用(特别是免疫原性)降至最低，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，达到治疗效果所需的剂量就越低，剂量的减少可以降低整体免疫反应。因此，我们将专注于对(-)-可卡因具有更高催化效率的CoCE突变体的合理设计、发现和临床前测试。要合理设计针对(-)-可卡因的高活性可卡因突变体，需要对可卡因催化水解的机理有详细的了解。这一机制可以通过使用最先进的分子建模、模拟和计算计算技术进行计算研究来理解。具体目标包括：(1)通过进行量子力学(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CoCE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。(2)通过使用最近发展的基于过渡态建模和模拟的新的计算设计方法来设计、发现和测试对(-)-可卡因具有更高催化效率的CoCE突变体，以计算评估大量假想的CoCE突变体，随后进行湿实验测试，包括定点突变、蛋白质表达和纯化，并进行体外和体内活性试验。这项研究的长期目标将是最终开发一种使用高活性Coce突变体的高效抗可卡因药物。公共卫生相关性：通过注射可卡因酯酶(Coce)来促进可卡因代谢已被认为是治疗可卡因过量和成瘾的一种有前途的策略。在这个项目中设计和发现的Coce的高活性突变体最终将导致一种有效的抗可卡因药物。