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一种药物治疗可卡因滥用。具体地说，本提案将研究细菌可卡因酯酶的耐热突变体的潜力，用于治疗可卡因滥用。可卡因滥用在美国是一个普遍存在的问题。长期使用可卡因会导致严重的身体和社会后果，但目前还没有fda批准的针对可卡因滥用的具体药物疗法。这项研究的重点是细菌蛋白可卡因酯酶（CocE），它是从一种红球菌细菌中分离出来的，这种细菌生活在古柯植物周围180摄氏度的土壤中。CocE能迅速水解可卡因，比正在开发的用于可卡因滥用和毒性的类似酶快得多。该实验室先前的研究表明，小剂量的cocE能够有效地阻断可卡因引起的死亡、抽搐、癫痫发作，以及血压、心率和QT间期的升高。尽管野生型CocE在体外370C的半衰期极短（13分钟），但一组新的突变L169K/G173Q使CocE的体外半衰期达到72小时。该突变体的半衰期延长和作用时间长，使其成为比野生型CocE更好的滥用治疗候选者。在野生型CocE以及其他可卡因水解分子方面的经验，使该小组独特地定位于推进这种CocE的耐热突变体的发展，以治疗可卡因滥用。基于细菌CocE的长效突变（L169K/G173Q）可能是一种有效的潜在治疗可卡因成瘾的假设，我们提出了两个具体目标：1)确定L169K/G173Q CocE对体内可卡因致死效应和强化效应的疗效。将评估L169K/G173Q CocE和野生型CocE对美国国立卫生研究院瑞士小鼠可卡因致死性的效价和作用时间。L169K/G173Q CocE对可卡因增强性能的影响将在大鼠可卡因自我给药模型中进行评估。2)测定L169K/G173Q CocE在体内的稳定性和活性。通过分析L169K/G173Q和野生型CocE的血浆半衰期、体内催化率、体外活性和免疫原性来检测酶的稳定性。我们希望开发一种耐热突变型细菌可卡因酯酶，用于可卡因滥用的药物治疗，这是一个广泛存在的社会问题，目前没有特定的FDA批准的治疗方法。耐热性可卡因酯酶能迅速将可卡因转化为无活性产物，从而消除或大大降低可卡因强烈的精神和生理作用。

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.