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

描述(申请人提供)：这项研究的长期目标是开发一种治疗可卡因滥用的药物疗法。具体地说，这项提案将审查细菌可卡因酯酶的耐热突变作为可卡因滥用治疗的可能性。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批准的治疗可卡因滥用的具体药物。这项研究的重点是细菌蛋白可卡因酯酶(CoE)，从生活在古柯植物周围180℃土壤中的红球菌中分离出来。Coce快速水解可卡因的速度比正在开发的可卡因滥用和毒性方面的类似酶快得多。该实验室以前所做的工作表明，小剂量的Coce能够有效地阻止可卡因引起的死亡、抽搐、癫痫以及血压、心率和QT间期的增加。虽然野生型在体外370℃(13分钟)有极短的半衰期，但一组新的突变L169K/G173Q使Coce的体外半衰期为72小时。这种突变体的半衰期延长和持续时间长，使其比野生型Coce更适合用于滥用治疗。在野生型Coce以及其他可卡因水解物分子Unquley方面的经验使该小组能够推动这种Coce耐热突变体的开发，走向治疗可卡因滥用的方向。基于细菌Coce的长效突变(L169K/G173Q)可能是一种有效的治疗可卡因成瘾的假设，提出了两个具体的目标：1)在体内确定L169K/G173Q Coce对可卡因的致死和增强作用的有效性。将评估L169K/G173Q Coce和野生型Coce对抗可卡因诱导的瑞士小鼠死亡的效力和持续时间。L169K/G173Q Coce对可卡因增强特性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的体内稳定性和活性。通过分析L169K/G173Q和野生型CoCE的血浆半衰期、体内催化活性、体外活性和免疫原性来考察酶的稳定性。我们希望将一种耐热突变的细菌可卡因酯酶开发成治疗可卡因滥用的药物疗法，可卡因滥用是一个广泛存在的社会问题，目前还没有FDA批准的具体治疗方法。耐热可卡因酯酶可迅速将可卡因转化为非活性产物，从而消除或极大地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.