RAPID DETOXIFICATION OF COCAINE BY BUTYRYLCHOLINESTERASE

丁酰胆碱酯酶对可卡因的快速解毒

• 批准号: 2593410
• 负责人: OKSANA LOCKRIDGE
• 金额: $ 17.13万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2593410
• 关键词: CHO cells; active sites; blood chemistry; cholinesterases; cocaine; detoxification; drug abuse chemotherapy; enzyme activity; enzyme mechanism; genetic manipulation; genetic susceptibility; genotype; human genetic material tag; human tissue; isozymes; laboratory rat; mutant; nonhuman therapy evaluation; polymerase chain reaction; postmortem; protein purification; recombinant proteins

DESCRIPTION (Applicant's Abstract): Cocaine has toxic effects on the brain and heart causing seizures, cerebral hemorrhage, a marked increased in heart rate, disturbance of heart rhythm, and hypertensive crisis. Death can occur even in young people. It is estimated that 6 million Americans are regular users of cocaine and that emergency rooms annually treat 100,000 patients for cocaine related problems. Treatment with purified human butyryl cholinesterase (EC 3.1.1.8) protected rats from cocaine-induced convulsions, lethality, cardiac arrhythmias, hypertension, and hyperactivity. Butyryl cholinesterase is the major detoxifying enzyme of the pharmacologically active cocaine isomer, (-)-cocaine. Butyryl cholinesterase hydrolyzes (-)-cocaine at a rate of 4 molecules of cocaine per molecule of enzyme per minute (kcat=4 per min). This rate is slow compared to the rate at which butyryl cholinesterase hydrolyzes the pharmacologically inactive cocaine isomer, (+)-cocaine, where kcat=10,000 per min. Our goal is to genetically engineer human butyryl cholinesterase to enable it to hydrolyze (-) cocaine at a rate approaching the rate at which it hydrolyzes (+)-cocaine. The difference between (-) and (+) cocaine is the position of a methyl ester group. It is expected that fitting this small group into the active site will require mutation of a few amino acids in the active site gorge. Mutants made with the polymerase chain reaction will be expressed in Chinese hamster ovary cells. The secreted enzymes will be purified and assayed for cocaine hydrolase activity. The enzyme with the highest kcat and highest binding affinity will be tested in rats to measure how much of the enzyme is needed to provide full protection from cocaine-induced toxicity, and to measure the efficiency with which it reverses cocaine toxicity. The product of this research, a human butyryl cholinesterase that rapidly hydrolyzes (-)-cocaine, has the potential to be useful for treating cocaine intoxicated patients. A final goal is to test the hypothesis that people who have genetic variants of butyryl cholinesterase are more susceptible to the toxic effects of cocaine. Test tube experiments have shown that the atypical variant (D70G) of butyryl cholinesterase has a 30 fold decrease in binding affinity for cocaine. This predicts that people with the atypical variant will react to a standard dose of cocaine as if it were an overdose, similar to their abnormal response to the muscle relaxant succinylcholine. To test this hypothesis, it is proposed to genotype the butyryl cholinesterase of people who have died after using cocaine. It is expected that butyryl cholinesterase genetic variants will be present in a higher frequency in cocaine-related fatalities.

描述（申请人的摘要）： 可卡因对大脑和心脏的毒性作用，导致癫痫发作，大脑 出血，心率明显增加，心律障碍， 和高血压危机。 即使在年轻人中也可能发生死亡。 这是 估计有600万美国人是可卡因的常规使用者， 急诊室每年治疗100,000名与可卡因有关的患者 问题。 用纯化的人丁酰基胆碱酯酶治疗（EC 3.1.1.8） 保护大鼠免受可卡因引起的抽搐，致死性，心脏 心律不齐，高血压和多动症。 丁酰基胆碱酯酶是 药理活性可卡因异构体的主要排毒酶， （-）-可卡因。 丁酰基胆碱酯酶水解（ - ） - 可卡因的速率为4 每分钟酶的可卡因分子每分钟（KCAT = 4分钟）。 与丁酰基胆碱酯酶的速率相比，此速率很慢。 水解药理学无活性的可卡因异构体，（+） - 可卡因，其中 KCAT =每分钟10,000。 我们的目标是基因工程人类的丁酰基 胆碱酯酶使其能够以接近的速率水解（ - ）可卡因 它水解（+） - 可卡因的速率。 （ - ）和 （+）可卡因是甲基酯基的位置。 预计 将这小组安装到活动位置中将需要一些突变 活动现场峡谷中的氨基酸。 用聚合酶制成的突变体 链反应将在中国仓鼠卵巢细胞中表达。 这 分泌的酶将被纯化并分析可卡因水解酶 活动。 具有最高KCAT和最高结合亲和力的酶 将在大鼠中进行测试，以测量需要多少酶 提供完全保护可卡因诱导的毒性，并测量 它逆转可卡因毒性的效率。 这个产品 研究，一种迅速水解的人丁酰基胆碱酯酶 （ - ） - 可卡因，有可能对可卡因陶醉有用 患者。 最终目标是检验具有遗传变异的人的假设 丁酰基胆碱酯酶更容易受到 可卡因。 试管实验表明，非典型变体（D70G） 丁酰基胆碱酯酶的结合亲和力降低了30倍 可卡因。 这预示着具有非典型变体的人会反应 可卡因的标准剂量，好像是用药过量一样，类似于它们 对肌肉松弛剂的异常反应。 测试这个 假设，提出了基因型的人丁酰基胆碱酯酶 使用可卡因后死亡的人。 预计丁Yesryl 胆碱酯酶遗传变异将以较高的频率存在 与可卡因有关的死亡。