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有毒性作用，导致癫痫发作、脑 出血、心率明显加快、心律紊乱、 和高血压危机。 即使是年轻人也可能发生死亡。 这是 据估计，有 600 万美国人经常吸食可卡因 急诊室每年治疗 100,000 名可卡因相关患者 问题。 用纯化的人丁酰胆碱酯酶（EC 3.1.1.8）处理 保护大鼠免受可卡因引起的惊厥、致死性、心脏病 心律失常、高血压和多动症。 丁酰胆碱酯酶是 药理活性可卡因异构体的主要解毒酶， （-）-可卡因。 丁酰胆碱酯酶以 4 的速率水解 (-)-可卡因 每分钟每分子酶的可卡因分子数（kcat=4 每分钟）。 与丁酰胆碱酯酶的速率相比，该速率较慢 水解药理学上无活性的可卡因异构体，(+)-可卡因，其中 kcat=10,000/分钟。 我们的目标是对人类丁酰进行基因改造 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 (-) 之间的区别 (+)可卡因是甲酯基团的位置。 预计 将这个小群体放入活性位点需要一些突变 活性位点峡谷中的氨基酸。 用聚合酶制成的突变体 连锁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检测可卡因水解酶 活动。 kcat 最高、结合亲和力最高的酶 将在老鼠身上进行测试，以测量需要多少酶 提供全面的保护，防止可卡因引起的毒性，并测量 它逆转可卡因毒性的效率。 这个产品的 研究发现，一种快速水解的人丁酰胆碱酯酶 (-)-可卡因，有可能用于治疗可卡因中毒 患者。 最终目标是检验具有遗传变异的人的假设 丁酰胆碱酯酶更容易受到以下物质的毒性作用 可卡因。 试管实验表明非典型变体（D70G） 丁酰胆碱酯酶的结合亲和力降低了 30 倍 可卡因。 这预示着携带非典型变异的人会对 标准剂量的可卡因，就像过量服用一样，类似于他们的 对肌肉松弛剂琥珀胆碱的异常反应。 为了测试这个 假设，建议对人的丁酰胆碱酯酶进行基因分型 使用可卡因后死亡的人。 预计丁酰 胆碱酯酶遗传变异在以下人群中的出现频率会更高 可卡因相关死亡事件。