RAPID DETOXIFICATION OF COCAINE BY BUTYRYLCHOLINESTERASE

描述（申请人摘要）： 可卡因对大脑和心脏有毒性作用，引起癫痫发作， 出血，心率明显加快，心律紊乱， 和高血压危象 即使在年轻人中也可能发生死亡。 是 据估计，有600万美国人经常吸食可卡因， 急诊室每年治疗10万名与可卡因有关的患者 问题 用纯化的人丁酰胆碱酯酶处理（EC 3.1.1.8） 保护大鼠免受可卡因诱导的惊厥、致死性、心脏 心律失常高血压和多动症 丁酰胆碱酯酶是 活性可卡因异构体的主要解毒酶， - 可卡因。 丁酰胆碱酯酶水解（-）-可卡因的速率为4 分子可卡因/分子酶/分钟（kcat=4/分钟）。 这个速率与丁酰胆碱酯酶 水解无活性的可卡因异构体（+）-可卡因，其中 kcat= 10，000每分钟。我们的目标是基因工程人类丁酰 胆碱酯酶，使其能够以接近 其水解（+）-可卡因的速率。 （-）和（-）之间的区别 （+）可卡因是甲酯基的位置。 预计在 将这个小基团装配到活性位点将需要几个突变。 活性位点峡谷中的氨基酸 用聚合酶制造的突变体 链反应将在中国仓鼠卵巢细胞中表达。 的 分泌的酶将被纯化并测定可卡因水解酶 活动 具有最高kcat和最高结合亲和力的酶 将在大鼠身上进行测试，以测量需要多少酶才能 提供对可卡因诱导的毒性的充分保护，并测量 它逆转可卡因毒性的效率。 这个产品 研究，一种人类丁酰胆碱酯酶， （-）-可卡因，具有用于治疗可卡因中毒的潜力 患者 最后一个目标是检验一个假设，即有基因变异的人 丁酰胆碱酯酶的毒性作用更敏感 可卡因 试管实验表明，非典型变异体（D 70 G） 丁酰胆碱酯酶的结合亲和力降低30倍， 可卡因 这预示着非典型变异的人会对 一个标准剂量的可卡因，如果它是过量，类似于他们的 对肌肉松弛剂琥珀胆碱的异常反应 为了验证这一 假设，提出对人的丁酰胆碱酯酶进行基因分型 吸食可卡因后死亡的人 预期丁酰基 胆碱酯酶遗传变异将以更高的频率存在于 与可卡因有关的死亡

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.