A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse
A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse
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DOI：
10.1124/mol.109.060806
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发表时间：
2010-04-01
影响因子：
3.6
通讯作者：
Woods, James H.
中科院分区：
文献类型：
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作者：
Brim, Remy L.;Nance, Mark R.;Woods, James H.
罗氏可卡因酯酶(CoCE)是治疗可卡因过量和可卡因成瘾的一种有吸引力的潜在疗法。Coce直接将可卡因降解成非活性产物，而传统的小分子方法需要阻断可卡因对各种单胺转运体和离子通道的抑制作用。野生型(Wt)可卡因酯酶的有效性因其在37℃下的失活而受到阻碍。在此，我们鉴定了迄今为止最耐热的可卡因酯酶形式Coce-L169K/G173Q。体外动力学分析表明，Coce-L169K/G173Q在37℃下的半衰期为2.9天，比wt提高了340倍，比以前报道的突变体高出15倍。在1.6埃分辨率下测定的CoCE-L169K/G173Q的结晶学分析表明，稳定化包括增强的磁区-磁区相互作用，包括van der Waals相互作用和氢键。活体啮齿动物研究表明，在小鼠静脉注射CoCE-L169K/G173Q之前，比wt Coce提供更长时间的保护，使其免受可卡因诱导的死亡。此外，静脉给药(预处理)Coce-L169K/G173Q以时间依赖的方式阻止可卡因的自我给药。CoCE的体内效应的终止似乎依赖于其从血浆中的清除，而不是成正比，因为它的半衰期约为2.3h，与wt Coce(2.2h)相似。综上所述，这些数据表明Coce-L169K/G173Q具有治疗可卡因滥用的生物疗法的许多特性，但需要进一步开发以改善其血清半衰期。
Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 degrees C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 degrees C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-angstrom resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.