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: 7506455
• 负责人: DONALD W LANDRY
• 金额: $ 49.89万
• 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/7506455
• 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; Numbers; Overdose; Performance; Pharmaceutical Preparations; Preclinical Testing; Process; Protocols documentation; Public Health; Reaction; Rodent Model; Roentgen Rays; Site-Directed Mutagenesis; Structure; Testing; Toxic effect; addiction; base; cocaine esterase; computer studies; design; esterase; immunogenicity; improved; in vivo; models and simulation; molecular dynamics; molecular modeling; mutant; novel; preclinical study; prevent; protein expression; quantum; response; therapeutic effectiveness; three dimensional structure

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）：通过施用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有前途的治疗策略。酯酶 CocE 是迄今为止已确定的代谢天然存在的 (-)-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，并且它有可能开发成化学上有用的拮抗剂，用于 (-)- 可卡因的毒性和行为影响可卡因。为了优化这种潜在的抗可卡因药物的功效并尽量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，达到治疗效果所需的剂量就越低，并且剂量的减少可以降低总体免疫反应。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，以提高对 (-)-可卡因的催化效率。针对 (-)-可卡因的 CocE 高活性突变体的合理设计需要详细了解 CocE 催化可卡因水解的机制。这种机制可以通过使用最先进的分子建模、模拟和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，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谱。和分子动力学（MD）模拟等。（2）通过使用最近开发的新型计算设计方法，设计、发现和测试对（-）-可卡因具有改进的催化效率的CocE突变体利用过渡态建模和模拟来计算评估大量假设的 CocE 突变体，然后进行湿实验测试，包括定点诱变、蛋白质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是最终利用 CocE 的高活性突变体开发一种有效的抗可卡因药物。公共卫生相关性：通过施用可卡因酯酶 (CocE) 增强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治疗策略。该项目中将设计和发现的 CocE 高活性突变体最终将产生一种有效的抗可卡因药物。