Molecular Evolution of Human Cocaine Catalysts

描述（由申请人提供）：可卡因滥用是美国的一个主要医疗和公共卫生问题，每年有200多万慢性、铁杆使用者和80，000例可卡因相关急诊。对社会造成的人身伤害和经济损失是巨大的。不幸的是，尽管进行了大量的工作，但目前还没有临床上有用的药物可用于有效治疗可卡因成瘾。迫切需要开发一种有效的药物，防止可卡因的行为和精神作用。一种有希望的替代方法是通过施用可卡因代谢酶，在可卡因到达大脑之前加速其在血液中分解为无害产物。我们在此提出使用强大的分子进化策略和高通量表达和功能筛选系统来显著改善可卡因催化剂的催化活性，以实现治疗效用所需的效力。具体目标包括：1）使用可卡因类似物构建高通量功能筛选平台; 2）通过筛选高度多样化的突变文库，体外进化候选酶，以改善可卡因解毒催化作用; 3）检查进化酶的生化功能。这项工作将提供通过多学科研究的组合方法确定可卡因催化酶所需的初步研究和开发工作。由于靶向酶已经被开发用于其他临床用途，如果进化工作证明是成功的，我们将有下一代可卡因催化剂准备进入临床前测试和先进的产品开发。本文提出的治疗剂的开发将为可卡因成瘾的管理提供显着的改善。我们建议使用强大的分子进化策略来创建可卡因水解酶，可以加速可卡因在血液中分解为无害的产物，以预防神经和心血管并发症。这项工作的成功将导致开发新的，安全的，有效的医疗对策，紧急戒毒的可卡因过量，并为慢性减少可卡因加强精神活性的影响。

DESCRIPTION (provided by applicant): Cocaine abuse is a major medical and public health concern in the United States with more than 2 million chronic, hardcore users and 80,000 cocaine-related ER visits annually. The personal damage and economic harm to society is enormous. Unfortunately, despite extensive work, no clinically useful agent is currently available to effectively treat cocaine addiction. An urgent need exists to develop an effective agent that prevents the behavioral and psychoactive effects of cocaine. One promising alternative approach is to accelerate the breakdown of cocaine to non-harmful products in blood before it reaches the brain by administration of cocaine-metabolizing enzymes. We propose herein to use powerful molecular evolution strategies and high throughput expression and functional screening systems to significantly improve the catalytic activity of cocaine catalysts to achieve the potency required for therapeutic utility. The Specific Aims includes 1) Validate high throughput functional screening platforms using a cocaine analogue; 2) In vitro evolution of candidate enzymes for improved cocaine detoxification catalysis by screening highly diversified mutation libraries; and 3) Examine biochemical functions of evolved enzyme. This work will provide the initial research and development effort required to identify cocaine catalytic enzymes through a combinatorial approach of multi-disciplinary research. Since the targeted enzyme has already been developed for other clinical use, we will have the next generation of cocaine catalysts ready to enter preclinical testing and advanced product development if the evolution effort proved successful. Development of the therapeutic agent proposed here will provide significant improvement for the management of cocaine addiction. We propose to use powerful molecular evolution strategies to create cocaine hydrolyzing enzymes that can accelerate the breakdown of cocaine to non-harmful products in blood to prevent neurological and cardiovascular complications. Success of this work will led to the development of novel, safe, and effective medical countermeasures for both emergency detoxification of cocaine overdose, and for chronic reduction of cocaine reinforcing psychoactive effect.