Rapid and responsive development of "spice" sensors using a novel recognition scaffold

使用新型识别支架快速响应地开发“香料”传感器

• 批准号: 10372178
• 负责人: Sean Cutler
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372178
• 关键词: Address; Affinity; Agonist; Analytical Chemistry; Back; Binding; Biological; Biological Assay; Biology; Black race; Cannabinoids; Cannabis; Cessation of life; Chemicals; Chimeric Proteins; Classification; Clinical; Complement; Complex; Coupled; Couples; Dangerousness; Data; Dendroaspis; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Dimerization; Directed Molecular Evolution; Discrimination; Drug Controls; Drug Screening; Drug abuse; Ensure; Enzymes; Evaluation; Evolution; Forensic Medicine; Goals; Health; Hormones; Hospitalization; Human; Immunoassay; Intoxication; Laboratories; Legal; Ligand Binding; Ligands; Luciferases; Mediating; Methods; Names; Output; Pharmacology; Phosphoric Monoester Hydrolases; Plants; Protein Fragment; Proteins; Psychoses; Reagent; Reporting; Research Personnel; Schedule; Signal Transduction; Specimen; Spices; Stress; Substance of Abuse; System; Techniques; Technology; Testing; Training; Work; analog; antibody detection; assay development; base; cannabinoid receptor; cost; design; detection platform; detection sensitivity; diagnostic assay; diagnostic tool; drug testing; experimental study; genetic selection; improved; innovation; marijuana use; mutant; new technology; novel; novel diagnostics; protein phosphatase 2C; rapid test; receptor; response; scaffold; sensor; small molecule; synthetic cannabinoid; tool

PROJECT SUMMARY Over the past decade, synthetic cannabinoids that intend to mimic the effects of cannabis use have emerged as an important group of New psychoactive substances (NPSs) sold as “legal high” products under brand names such as “Spice”, “Black Mamba”, and “Annihilation”. These compounds are more potent and dangerous agonists than those found in cannabis and they have caused a variety of adverse health effects, including psychoses, hospitalizations, and deaths. In response, the DEA has placed 43 synthetic cannabinoids under Schedule I classification; however, new synthetic cannabinoid ingredients are regularly created and released to both evade legal restrictions and provide a product that avoids detection by routine drug screening tests. Although GC/LC-MSn can be used to comprehensively detect these agents, the vast majority of forensic and clinical labs rely on immunoassays for their drug tests due to simplicity and cost. New methods that accelerate the development of diagnostic for the ever-changing substances of abuse would be useful, both for addressing the synthetic cannabinoid problem and as a general technology. We propose to address this challenge by developing a new platform for rapid assay development that functions orthogonally to immunoassays. To do this, we propose to use directed evolution to reprogram the ligands sensed by the plant PYR1-PP2C stress hormone sensing system. This sensor functions through a naturally occurring chemical-induced dimerization (CID) mechanism that couples ligand recognition by PYR1 to the formation of a stable PYR1-PP2C complex; this feature facilitates genetic selection experiments for receptors that recognize new ligands. We hypothesize that our platform will enable rapid development cycles for new diagnostics. To prove this hypothesis, this exploratory study will (1) Develop receptors for selective recognition of synthetic cannabinoids through directed evolution, (2) Integrate target recognition and signal output using protein fragment complementation assays, and (3) Establish and validate synthetic cannabinoid detection systems in biological matrices. Sensors will be designed to detect multiple synthetic cannabinoids, prioritizing the most prevalent synthetic cannabinoid of 2020 (5F-MDMB-PICA), which is not detected by current clinical immunoassays. The feasibility of this proposal is backed by extensive preliminary data from the PI’s laboratory, which demonstrates that the PYR1-PP2C system can be used to evolve high-affinity synthetic cannabinoid sensors. Our long-term goal is to establish this new system for the rapid development of diagnostic tools and deliver reagents that enable fast and selective detection of synthetic cannabinoids in low volumes of biological specimens. These enabling tools will be valuable for biomedical and clinical analyses of synthetic cannabinoids to control drug abuse, improve treatment and diagnosis of intoxication, to characterize their pharmacological and health effects, and as a general platform for rapidly evolving new diagnostic assays.

项目摘要 在过去的十年中，出现了旨在模仿大麻使用效果的合成大麻素 作为一组重要的新精神活性物质（NPSs），以“法律的高”品牌产品销售 比如“香料”、“黑曼巴”和“毁灭”。这些化合物的毒性更强，更危险 兴奋剂比大麻中发现的兴奋剂，它们引起了各种不利的健康影响，包括 精神病住院和死亡作为回应，DEA已将43种合成大麻素置于 附表I分类;然而，新的合成大麻素成分定期产生和释放， 两者都避开了法律的限制，并提供了避免通过常规药物筛选试验检测的产品。 虽然GC/LC-MSn可用于全面检测这些试剂，但绝大多数法医和 由于简单和成本，临床实验室依赖免疫测定法进行药物测试。新方法加速 开发针对不断变化的滥用物质的诊断方法将是有益的， 合成大麻素的问题，并作为一种通用技术。我们建议通过以下方式应对这一挑战： 开发一个新的平台，用于快速检测开发，其功能与免疫检测正交。做 因此，我们建议使用定向进化来重编程植物PYR 1-PP 2C胁迫感受的配体 激素感应系统该传感器通过自然发生的化学诱导的二聚作用发挥作用 (CID)将PYR 1的配体识别与稳定的PYR 1-PP 2C复合物的形成偶联的机制; 该特征有利于识别新配体的受体的遗传选择实验。我们假设 我们的平台将为新诊断技术提供快速开发周期。为了证明这一假设， 探索性研究将（1）通过以下途径开发选择性识别合成大麻素的受体： （2）利用蛋白质片段将目标识别和信号输出结合起来 互补测定，和（3）建立和验证合成大麻素检测系统， 生物基质传感器将被设计为检测多种合成大麻素，优先考虑最 2020年流行的合成大麻素（5 F-MDMB-PICA），目前的临床试验未检测到 免疫测定。这项建议的可行性得到了PI实验室大量初步数据的支持， 这表明PYR 1-PP 2C系统可用于进化高亲和力的合成大麻素 传感器.我们的长期目标是建立这个新系统，以快速开发诊断工具， 提供能够快速和选择性检测低体积生物制品中的合成大麻素的试剂， 标本这些使能工具将对合成药物的生物医学和临床分析有价值。 大麻素控制药物滥用，改善中毒的治疗和诊断， 药理学和健康影响，并作为一个通用平台，快速发展新的诊断分析。