Advancing the therapeutic potential of a nicotine-degrading enzyme

项目概要/摘要。烟草成瘾是世界上第二大死亡原因， 癌症和心脏病的最大病因根据目前的吸烟趋势，烟草使用将导致1000万人死亡。 到2020年，每年。每年，4200万成年吸烟者中有近一半试图戒烟;然而，由于 尼古丁具有高度成瘾性，只有不到5%的人成功。帮助戒烟包括支持 减少尼古丁奖赏和戒断的咨询、尼古丁替代和受体拮抗剂 症状不幸的是，尼古丁替代疗法的长期结果仍然很差， 第一年后的戒烟率只有10-20%。滥用药物疫苗诱导抗体， 尼古丁等药物的药理作用。到目前为止，戒烟疫苗已经显示出希望， 临床前动物模型，以证明其减少尼古丁介导的生理和行为反应的能力。 然而，在临床研究中，这些疫苗未能衡量戒烟的显着差异 在干预组和安慰剂组之间。尼古丁疫苗的致命弱点是它们不能 持续产生高抗体浓度以减少循环游离药物。一种生物体， 分解尼古丁，而不是简单地隔离药物，可以克服尼古丁的缺点， 疫苗这项拟议中的研究详细介绍了一种细菌菌株，恶臭假单胞菌，它已经进化到使用尼古丁 作为其唯一的碳和氮来源。从这种细菌菌株中分离出一种一流的酶， 尼古丁氧化还原酶（NicA 2），其在将尼古丁降解为非精神活性产物方面是高效的。 酶的表征表明它是尼古丁戒烟治疗的极好候选者。然而， 这种策略的成功证明将需要如具体实施例中所概述的酶的改进。 目的包括：（1）工程化NicA 2以增加其在血清中的稳定性并降低免疫原性倾向。 (2)评估NicA 2对尼古丁在非依赖性和依赖性中的增强特性的影响 啮齿动物模型对烟草使用障碍具有高预测有效性。我的建议将采取现有的 酶的策略，用于衰减尼古丁的精神活性的影响，并工程师一个临床可行的 尼古丁戒断治疗剂。

Project Summary/Abstract. Tobacco addiction is the second leading cause of death in the world and the single largest cause of cancer and heart disease. Given present smoking trends, tobacco use will kill 10 million people each year by 2020. Each year nearly half of the 42 million adult smokers attempt to quit; yet, due to the highly addictive nature of nicotine, less than 5% succeed. Aids to smoking cessation include supportive counseling, nicotine replacement and receptor antagonists that reduce nicotine reward and withdrawal symptoms. Unfortunately, long-term outcomes for nicotine replacement therapies remain poor and achieve an abstinence rate of only 10-20% after the first year. Drugs of abuse vaccines induce antibodies that block the pharmacological effects of drugs like nicotine. To date, vaccines for smoking cessation have shown promise in preclinical animal models for their ability to diminish nicotine-mediated physiological and behavioral responses. However, in clinical studies these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. The Achilles' heel of nicotine vaccines has been their inability to consistently generate high antibody concentrations to reduce circulating free drug. A biologic capable of catabolizing nicotine, rather than simply sequestering the drug, could overcome the shortcomings of a nicotine vaccine. The proposed study details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain, was isolated a first-in-class enzyme, a nicotine oxidoreductase (NicA2), that is highly efficient at degrading nicotine to a non-psychoactive product. Characterization of the enzyme indicates it to be an excellent candidate for nicotine cessation therapy. Yet, successful demonstration of such a strategy will require improvement of the enzyme as outlined in specific aims including: (1) Engineering NicA2 to increase its stability in serum and reduce immunogenicity liabilities. (2) Evaluating the effect of NicA2 on the reinforcing properties of nicotine in non-dependent and dependent rodent models with high predictive validity for tobacco use disorder. My proposal will take an existing enzymatic strategy for the attenuation of nicotine's psychoactive effects and engineer a clinically viable therapeutic for nicotine cessation.