Towards the Treatment of Cocaine Abuse with a Mutant Bacterial Cocaine Esterase

描述（由申请人提供）：本研究的长期目标是开发可卡因滥用的药物治疗。具体来说，该提案将研究细菌可卡因酯酶的热稳定突变体用作可卡因滥用治疗的潜力。滥用可卡因在美国是一个普遍的问题。长期使用可卡因会导致严重的身体和社会后果，但目前没有FDA批准的针对可卡因滥用的药物治疗。这项研究的重点是细菌蛋白可卡因酯酶（CocE），分离自红球菌细菌，生活在180 ℃的土壤周围的古柯植物。CocE快速水解可卡因的速度明显快于正在开发的用于可卡因滥用和毒性的类似酶。该实验室以前的工作表明，小剂量的cocE能够有效地阻止可卡因引起的死亡，惊厥，癫痫发作以及血压，心率和QT间期的增加。虽然野生型形式在体外370 ℃下具有极短的半衰期（13分钟），但一组新的突变L169 K/G173 Q使CocE的体外半衰期为72小时。该突变体的延长的半衰期和长的作用持续时间使其成为比野生型CocE更好的滥用治疗剂的候选者。野生型CocE以及其他可卡因水解分子的经验使这一小组能够推进CocE的这种热稳定突变体的发展，以治疗可卡因滥用。 基于细菌CocE的长效突变（L169 K/G173 Q）可能是一种有效的潜在治疗可卡因成瘾的假设，提出了两个具体目标：1）确定L169 K/G173 Q CocE对可卡因的体内致死和增强效应的效力。将评估L169 K/G173 Q CocE和野生型CocE对NIH Swiss小鼠中可卡因诱导的致死性的效力和作用持续时间。将在大鼠可卡因自我给药模型中评估L169 K/G173 Q CocE对可卡因的强化性质的影响。2)测定L169 K/G173 Q CocE的体内稳定性和活性。将通过分析L169 K/G173 Q和野生型CocE的血浆半衰期、体内催化速率、离体活性和免疫原性来检查酶稳定性。 我们希望开发一种热稳定的突变细菌可卡因酯酶，用于可卡因滥用的药物治疗，这是一个广泛的社会问题，目前还没有FDA批准的具体治疗方法。热稳定的可卡因酯酶迅速将可卡因转化为无活性产物，因此消除或大大降低了可卡因的强烈精神和生理作用。

DESCRIPTION (provided by applicant): The long term goal of this research is to develop a pharmacological treatment for cocaine abuse. Specifically, this proposal will examine the potential of a thermostable mutant of bacterial cocaine esterase to be used as a therapy for cocaine abuse. Cocaine abuse is a widespread problem in the United States. Chronic cocaine use leads to severe physical and social consequences yet currently there is no specific FDA-approved pharmacotherpay for cocaine abuse. This research focuses on the bacterial protein cocaine esterase (CocE), isolated from a Rhodococcus bacterium that lives in the 180C soil surrounding the coca plant. CocE rapidly hydrolyzes cocaine significantly faster than similar enzymes that are in development for cocaine abuse and toxicity. Previous work done by this lab has shown that small doses of cocE are able to effectively block cocaine-induced lethality, convulsions, seizure, as well as increases in blood pressure, heart rate and QT interval. Although the wild type form has an extremely short half-life at 370C in vitro (13 minutes), a new set of mutations, L169K/G173Q, give CocE an in vitro half life of 72 hours. The extended half-life and long duration of action of this mutant makes it a better candidate for an abuse therapeutic than wild-type CocE. Experience with wild-type CocE as well as other cocaine hydrolyzing molecules uniquley positions this group to advance the development of this thermostable mutant of CocE towards a therapy for cocaine abuse. Based on the hypothesis that the long acting mutation of bacterial CocE (L169K/G173Q) may be an effective potential treatment for cocaine addiction, two specific aims are proposed: 1) Determine the efficacy of L169K/G173Q CocE against the lethal and reinforcing effects of cocaine in vivo. The potency and duration of action of L169K/G173Q CocE and wild type cocE against cocaine-induced lethality in NIH Swiss mice will be assessed. The effects of L169K/G173Q CocE on the reinforcing properties of cocaine will be assessed in a rat cocaine self-administration model. 2) Determine the stability and activity of L169K/G173Q CocE in vivo. Enzyme stability will be examined by analyzing the plasma half-life, in vivo rates of catalysis, ex vivo activity and immunogenicity of L169K/G173Q and wild-type CocE. We hope to develop a thermostable mutant bacterial cocaine esterase into a pharmacotherapy for cocaine abuse, a widespread social problem for which there is currently no specific FDA approved treatment. Thermostable cocaine esterase rapidly converts cocaine into inactive products, therefore eliminating or greatly reducing the strong psychotropic and physiological effects of cocaine.