A Nicotine Biosensor for Addiction Studies

描述（由申请人提供）：与烟草有关的死亡每年使美国损失约2000亿美元。尼古丁是导致吸烟上瘾的主要原因，一旦进入人体，在15秒内迅速影响大脑。戒掉尼古丁成瘾是一项挑战，复发率仍然很高。尼古丁作用的极快时间尺度使得研究尼古丁在大脑中积累的现有技术（如微透析、PET、放射免疫测定）不适合在自由运动的动物中进行长期成瘾研究。生物传感器是监测中枢神经系统神经化学物质浓度实时变化的成熟技术。生物传感器最关键的组成部分是用作生物识别元素的酶，而生物传感器最终设计的任何方面都不如具有足够活性和稳定性的适当折叠酶更重要。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℃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。我们将结合结构引导设计和定向蛋白质进化（包括一个合适的选择过程）来磨砺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影响尼古丁成瘾的研究，它允许在长达一周的时间内实时记录自由运动动物的中枢神经系统尼古丁浓度。此外，尼古丁生物传感器可以与其他生物传感器结合使用，这将允许同时监测尼古丁浓度的变化与其他重要的中枢神经系统分析物（即葡萄糖，谷氨酸盐，乳酸盐）或成瘾化合物（即乙醇）串联。这种方法有望揭示尼古丁分布、动态和通量的新见解。此外，尼古丁生物传感器应该作为一种筛选工具，用于开发新的药物制剂，以帮助戒烟和抑制复发。I期和II期组件的完成将为科学界提供两个重要的创新：1)尼古丁生物传感器适用于长期（即长达一周）成瘾研究，可提供中枢神经系统中尼古丁浓度的逐秒变化。2)一种开发新型生物传感器的改进方法，该方法针对对成瘾很重要的分析物，并且目前不存在氧化酶（即可卡因和咖啡因）。

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.