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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要在过去的十年中，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型精神活性物质 (NPS)，以品牌下的“合法高”产品出售 “香料”、“黑曼巴”和“湮灭”等名称更有效、更危险。与大麻中发现的激动剂相比，它们对健康造成了多种不利影响，包括作为回应，DEA 将 43 种合成大麻素纳入管制范围。附表 I 分类；但是，新的合成大麻素成分会定期生产并释放给两者都规避了法律限制，并提供了避免常规药物筛选测试检测到的产品。尽管 GC/LC-MSn 可用于全面检测这些物质，但绝大多数法医和由于简单且成本较高的新方法，临床实验室依靠免疫分析进行药物测试。开发针对不断变化的滥用物质的诊断方法将非常有用，既可以解决合成大麻素问题并作为一项通用技术，我们建议通过以下方式解决这一挑战。开发一个新的快速检测开发平台，其功能与免疫检测正交。为此，我们建议使用定向进化来重新编程植物 PYR1-PP2C 胁迫感知的配体该传感器通过自然发生的化学诱导二聚作用发挥作用。 (CID) 将 PYR1 的配体识别与稳定的 PYR1-PP2C 复合物的形成耦合的机制；这一功能有利于识别新配体的受体的遗传选择实验。我们的平台将实现新诊断的快速开发周期为了证明这个假设，这。探索性研究将（1）开发选择性识别合成大麻素的受体定向进化，（2）利用蛋白质片段整合目标识别和信号输出互补测定，以及 (3) 建立并验证合成大麻素检测系统传感器将被设计用于检测多种合成大麻素，优先考虑最重要的。 2020年流行的合成大麻素（5F-MDMB-PICA），目前临床尚未检测到该提案的可行性得到了 PI 实验室的大量初步数据的支持，这表明PYR1-PP2C系统可用于进化高亲和力的合成大麻素我们的长期目标是建立这个新系统，以快速开发诊断工具和传感器。提供能够快速、选择性地检测少量生物样品中合成大麻素的试剂这些支持工具对于合成的生物医学和临床分析非常有价值。大麻素用于控制药物滥用、改善中毒的治疗和诊断、表征其药理和健康影响，并作为快速发展的新诊断测定的通用平台。