NOVEL COCAINE ESTERASES

本建议书中描述的工作是申请KO2的一部分 研究科学家发展奖。成人丁酰胆碱酯酶 是一种关键的血清酶，能分解和解毒许多外源物质。 含有羧酸酯部分的。虽然这种酶已经被 研究了很多年，是这个领域的起点 药物遗传学，令人惊讶的是，酶的生理作用是 未知。我们研究的长期目标是开发出更有效的 脱毒生物催化剂用于可卡因的水解和脱除 人体组织。这样的反可卡因催化剂可能会挽救生命。小才是 已知人类丁酰胆碱酯酶氨基酸突变的影响 可卡因的水解液。丁酰胆碱酯酶是胆碱酯酶的组成部分之一。 保护人类免受潜在有毒化学物质伤害的防御系统 他们的环境。丁酰胆碱酯酶含量降低的人类 活性可能易受外源物质的有毒化学作用的影响 包括滥用药物。 建议研究分为四大部分：L)采购 用于动力学和机理研究的人类丁酰胆碱酯酶变体， 2)丁酰胆碱酯酶作为脱毒催化剂的评价 用新的生物分析方法对可卡因进行水解，3)设计 丁酰胆碱酯酶变异体的分子模拟，以及4)表达 中国仓鼠卵巢(CHO)细胞中新的活性变异体。具体的 第2节的目的包括验证重组人的活性 酵素。第2节的具体目标包括对 通过新的高效液相色谱和放射分析检测变异型重组酶。这个 第三节的具体目的包括丁酰胆碱酯酶的研究。 使用分子建模和分子测量设备。具体目标 包括定点突变体的表达。 CHO细胞中的丁酰胆碱酯酶。我们将检测被诱变的 丁酰胆碱酯酶的催化活性。目前的结果是 研究将提供结构-功能的详细图景 人丁酰胆碱酯酶关系的研究。这项研究将提供一个 了解丁酰胆碱酯酶在代谢中作用的基础 可卡因。这项工作的意义在于它将导致更多的 深入了解丁酰胆碱酯酶在可卡因中的作用 以及其他滥用解毒的药物。

The work described in this proposal is part of a request for an KO2 Research Scientist Development Award. The adult human butyrylcholinesterase is a key serum enzyme that hydrolyzes and detoxicates many xenobiotics containing a carboxylic acid ester moiety. While the enzyme has been studied for many years and was the starting point for the field of pharmacogenetics, surprisingly, the physiological role of the enzyme is unknown. The longterm goal of our research is to develop more effective detoxication biocatalysts for the hydrolysis and removal of cocaine from human tissues. Such anti-cocaine catalysts could be life-saving. Little is known about the effect of human butyrylcholinesterase amino acid mutations on cocaine hydrolysis. Butyrylcholinesterase is one of the components of the defense that protects humans against potentially toxic chemicals in their environment. Humans with a decreased amount of butyrylcholinesterase activity may be predisposed to the toxic chemical action of xenobiotics including drugs of abuse. The proposed studies are divided into four major sections: l) procurement of human butyrylcholinesterase variants for kinetic and mechanism studies, 2) Evaluation of butyrylcholinesterase as a detoxication catalyst for cocaine hydrolysis by novel bioanalytical methods, 3) Design of butyrylcholinesterase variants with molecular modeling, and 4) Expression of new, active variants in Chinese Hamster Ovary (CHO) cells. The specific aims of section 2 include the verification of the activity of recombinant enzyme. The specific aims of section 2 include the kinetic evaluation of the variant recombinant enzymes by novel HPLC and radiometric assays. The specific, aims of section 3 include the study of butyrylcholinesterase using molecular modeling and molecular measuring devices. The specific aims of section 4 include the expression of site-directed mutants of butyrylcholinesterase in CHO cells. We will examine the mutagenized butyrylcholinesterases for catalytic activity. The results of the present study will provide a detailed picture of the structure-function relationships of human butyrylcholinesterase. The study will provide a basis for understanding a role of butyrylcholinesterase in the metabolism of cocaine. The significance of the work is that it will lead to a more sophisticated understanding of a role of butyrylcholinesterase in cocaine and other drugs of abuse detoxication.