A Nicotine Biosensor for Addiction Studies

烟草相关的死亡每年花费美国大约2000亿美元。尼古丁是吸烟的依赖性形成特性的唯一原因，一旦进入人体，它会在15秒内迅速影响大脑。打破尼古丁成瘾具有挑战性，复发率仍然很高。尼古丁作用的极快时间尺度使得现有的用于研究尼古丁在大脑中积累的技术（例如，微透析、PET、放射免疫测定）不适合在自由移动的动物中进行长期成瘾研究。生物传感器是一种成熟的技术，用于监测中枢神经系统神经化学物质浓度的实时变化。生物传感器最关键的组成部分是用作生物识别元件的酶，并且生物传感器的最终设计的任何方面都不如具有足够活性和稳定性特征的正确折叠的酶重要。到目前为止，还没有尼古丁特异性酶的报道。第一阶段SBIR提案的主要目标是开始将现有氧化酶的kcat、37 ℃下的稳定性、Km和Tm转化为能够特异性检测尼古丁的酶-这一过程将在第二阶段完成。我们将结合联合收割机结构导向设计与定向蛋白质进化（包括一个合适的选择过程），磨练和优化一种新的尼古丁氧化酶适合作为尼古丁生物传感器的基础。由此产生的尼古丁生物传感器有望通过允许在自由移动的动物中实时记录CNS尼古丁浓度长达一周来影响尼古丁成瘾研究。此外，尼古丁生物传感器可以与其他生物传感器结合使用，这将允许与其他重要的CNS分析物（即，葡萄糖、谷氨酸盐、乳酸盐）或成瘾化合物（即，乙醇）。这种方法有望揭示尼古丁分布，动力学和通量的新见解。此外，尼古丁生物传感器应发现用作筛选工具，用于开发新的药理学试剂，旨在帮助戒烟和抑制复发。第一阶段和第二阶段的完成将为科学界提供两个重要的创新：1）适合长期（即，长达一周）的成瘾研究，提供了中枢神经系统中尼古丁浓度的逐秒变化。2)一种用于开发新的生物传感器的改进方法，所述生物传感器靶向对成瘾重要的分析物并且目前不存在氧化酶（即，可卡因和咖啡因）。 公共卫生相关性：据估计，50%的经常吸烟者死于与吸烟有关的并发症。此外，与吸烟相关的尼古丁成瘾造成了巨大的经济损失，美国每年损失近2000亿美元，其中包括970亿美元的生产力损失和960亿美元的医疗保健支出。该项目可以更好地了解尼古丁成瘾，这可以促进戒烟研究，并可能在美国每年预防45万例与吸烟有关的死亡。

DESCRIPTION (provided by applicant): Tobacco-related deaths cost the United States approximately $200 billion each year. Nicotine is singularly responsible for the dependence-forming properties of tobacco smoking and, once introduced to the body, rapidly affects the brain within 15 seconds. Breaking nicotine addiction is challenging and relapse rates remain high. The extremely rapid timescale of nicotine action renders existing techniques for studying nicotine accumulation in the brain (e.g., microdialysis, PET, radioimmunoassay) ill-suited for long-term addiction studies in freely moving animals. Biosensors are a proven technology for monitoring real-time changes in CNS neurochemical concentrations. The most critical component of a biosensor is the enzyme used as the biorecognition element, and no aspect of a biosensor's final design is as vital as a properly folded enzyme with sufficient activity and stability profiles. To date, no nicotine-specific enzyme has been reported. A primary goal of this Phase I SBIR proposal is to begin the transformation of an existing oxidase enzyme's kcat, stability at 37oC, Km, and Tm into an enzyme capable of the specific detection of nicotine - a process that will be completed during Phase II. We will combine structure-guided design with directed protein evolution (including a suitable selection process) to hone and optimize a new nicotine oxidase enzyme suitable as the foundation of a nicotine biosensor. The resulting nicotine biosensor promises to impact nicotine addiction studies by allowing real-time recordings of CNS nicotine concentrations in freely moving animals for up to one week. Furthermore, the nicotine biosensor could be used in conjunction with other biosensors, which will allow for the simultaneous monitoring of changes in nicotine concentration in tandem with other important CNS analytes (i.e., glucose, glutamate, lactate) or addictive compounds (i.e., ethanol). This approach promises to reveal new insights into nicotine distribution, dynamics and flux. Furthermore, a nicotine biosensor should find use as a screening tool for the development of new pharmacologic agents designed to assist in smoking cessation and inhibit relapse. The completion of both Phase I and Phase II components will provide two important innovations to the scientific community: 1) A nicotine biosensor suitable for long-term (i.e., up to one week) addiction studies that provides second-by-second changes of nicotine concentration in the CNS. 2) A refined approach for the development of new biosensors that target analytes important for addiction and for which no oxidase enzyme currently exists (i.e., cocaine and caffeine). PUBLIC HEALTH RELEVANCE: It is estimated that 50% of regular smokers die due to smoking-related complications. In addition, nicotine addiction associated with cigarette smoking takes a tremendous economic toll, costing the United States nearly $200 billion annually, including $97 billion in lost productivity and $96 billion in health care expenditures. This projec allows for a better understanding of nicotine addiction, which could advance smoking cessation research and potentially prevent 450,000 smoking-related deaths per year in the United States.