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，导致癫痫发作，大脑 出血，心率显著增加，心律紊乱， 和高血压危机。甚至在年轻人身上也可能发生死亡。它是 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 有问题。纯化的人丁酰胆碱酯酶(EC 3.1.1.8)治疗 保护大鼠免受可卡因诱发的惊厥、致命性、心脏 心律失常、高血压和多动症。丁酰胆碱酯酶是 具有药理活性的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 每分子酶每分钟可卡因分子数(kcat=每分钟4个)。 与丁酰胆碱酯酶相比，这一速率较慢 对药理上不活跃的可卡因异构体(+)-可卡因进行水解，其中 Kcat=每分钟10,000。我们的目标是通过基因工程来改造人类丁酰基 胆碱酯酶，使其能够以接近的速度水解(-)可卡因 它水解(+)-可卡因的速度。(-)和(-)之间的区别 (+)可卡因是甲酯基团的位置。预计 将这一小群放入活性位点需要几个突变 活性部位峡谷中的氨基酸。用聚合酶产生的突变体 链式反应将在中国仓鼠卵巢细胞中表达。这个 将对分泌的酶进行纯化并检测可卡因水解酶 活动。具有最高kcat和最高结合亲和力的酶 将在老鼠身上进行测试，以测量需要多少酶来 提供针对可卡因引起的毒性的全面保护，并测量 它逆转可卡因毒性的效率。它的产物是 研究，一种能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 病人。 最终的目标是检验这样一种假设，即拥有基因变异的人 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(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种的人会对 一种标准剂量的可卡因，就像是过量一样，类似于他们的 对肌肉松弛药琥珀胆碱的异常反应。为了测试这一点 假设，建议对人的丁酰胆碱酯酶进行基因分型。 因吸食可卡因而死亡。预计丁酰基 胆碱酯酶基因变异将出现更高的频率 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.