Novel Therapeutic Approaches for Narcotic Overdose

描述(申请人提供)：可卡因和海洛因由肝脏中存在的羧酸酯酶(CE)代谢。在可卡因中，甲基被人羧酸酯酶1(HCE1)去除，生成苯基淫羊藿碱，这是该药物的主要非活性代谢物。对于海洛因，这两种甲酯被同一种酶顺序水解制得6-单乙酰吗啡，然后是吗啡。我们最近测定了hCE1的X射线晶体结构，分别与可卡因和海洛因类似物--同型马托品和纳洛酮--形成了络合物。利用这些信息，我们将在hCE1氨基酸序列中设计突变，以提高其水解可卡因的能力。这些酶将在酯酶缺乏的动物模型中进行测试，以确定它们是否可以用于清除血液中的可卡因。此外，我们将确定这些突变体在乙醇存在下交换可卡因生成可卡因的能力，并测量不同hCE1蛋白对该化合物的水解率。 因此，这项应用的具体目的是：1)产生具有更高的可卡因水解酶催化效率的hCE1突变体；2)确定hCE1参与可卡因与可卡因的酯交换反应生成可卡因乙烯的氨基酸残基；3)阐明可卡因和可卡因类似物中工程形式的hCE1的晶体结构；以及4)评估hCE1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，这些研究应该能够开发治疗可卡因过量的新的治疗方法。

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.