Novel Therapeutic Approaches for Narcotic Overdose

性状（由申请方提供）：可卡因和海洛因通过肝脏中的羧酸酯酶（CE）代谢。对于可卡因，甲基被人羧酸酯酶1（hCE 1）去除，产生苄基芽子碱，这是药物的主要非活性代谢物。对于海洛因，两种甲酯依次被相同的酶水解，产生6-单乙酰吗啡，随后产生吗啡。我们最近确定了hCE 1的X射线晶体结构，分别与可卡因和海洛因类似物后马托品和纳洛酮复合。利用这些信息，我们将在hCE 1氨基酸序列中进行突变，以提高其水解可卡因的能力。这些酶将在缺乏酯酶的动物模型中进行测试，以确定它们是否可以用于从血液中清除可卡因。此外，我们将确定这些突变体在乙醇存在下转化可卡因以形成可卡因乙烯的能力，并测量不同hCE 1蛋白对该化合物的水解。 因此，本申请的具体目的是：1）产生对可卡因水解具有改进的催化效率的hCE 1突变体; 2）确定参与hCE 1将可卡因转成可卡因乙烯的酯交换反应的氨基酸残基; 3）阐明与可卡因和可卡因类似物的复合物中的hCE 1的工程化形式的晶体结构;和4）评估hCE 1的突变形式对暴露于可卡因的血浆酯酶缺陷小鼠的影响。总体而言，这些研究应允许开发用于治疗可卡因过量的新治疗方法。

DESCRIPTION (provided by applicant): Cocaine and heroin are metabolized by carboxylesterases (CE) present in the liver. With cocaine, the methyl group is removed by human carboxylesterase 1 (hCE1) to produce benzyl ecgonine, the major inactive metabolite of the drug. With heroin, both methyl esters are sequentially hydrolyzed by the same enzyme to yield 6-monoacetylmorphine and subsequently, morphine. We have recently determined the x-ray crystal structure of hCE1, in complex with the cocaine and heroin analogs, homatropine and naloxone, respectively. Using this information, we will engineer mutations within the hCE1 amino acid sequence to improve its ability to hydrolyze cocaine. These enzymes will be tested in an esterase-deficient animal model to determine whether they can be used to purge cocaine from the blood stream. Additionally, we will determine the ability of these mutants to transesterifiy cocaine in the presence of ethanol to form cocaethylene, and measure the hydrolysis of this compound by different hCE1 proteins. The Specific Aims of this application are therefore to: 1) generate hCE1 mutants with improved catalytic efficiency for cocaine hydrolysis; 2) determine the amino acid residues involved in the transesterification reaction of cocaine to cocaethylene by hCE1; 3) elucidate crystal structures of engineered forms of hCE1 in complexes with cocaine and cocaine analogues; and 4) assess the impact of mutant forms of hCE1 on plasma esterase-deficient mice exposed to cocaine. Overall, these studies should allow development of novel therapeutic approaches for the treatment of cocaine overdose.