Multiplexed Measurement of Psychoactive Drug Selectivity Profiles

精神活性药物选择性谱的多重测量

• 批准号: 7671666
• 负责人: Peter Krutzik
• 金额: $ 15.04万
• 依托单位: PRIMITY, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7671666
• 关键词: Agonist; Aliquot; Arrestins; Binding; Biological; Biological Assay; CD19 gene; CD8B1 gene; Calcium; Cell Line; Cell surface; Cells; Chemicals; Complex Mixtures; Cyclic AMP; Data; Development; Disease; Drug abuse; Dyes; Endocytosis; Ensure; Environment; Evaluation; Flow Cytometry; Freezing; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; Generations; Generic Drugs; Genetic; Human; Inhibitory Concentration 50; Label; Ligand Binding; Ligands; Measurement; Measures; Methodology; Methods; Noise; Performance; Pharmaceutical Preparations; Pharmacology; Phase; Phytotherapy; Plant Extracts; Population; Psychotropic Drugs; Reading; Reagent; Receptor Activation; Reporting; Sampling; Screening procedure; Second Messenger Systems; Serum; Signal Transduction; Small Business Innovation Research Grant; Specificity; Stimulus; Surface; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Tube; Validation; base; cell type; cost; interest; novel; public health relevance; receptor; receptor internalization; response; second messenger

DESCRIPTION (provided by applicant): The subject of this Phase I SBIR proposal is the development of a screening system that quantifies the effects of candidate psychoactive drugs across 16 different GPCRs in a single sample. Many endogenous GPCR ligands target multiple receptors. Specific biological effects are often achieved through a "selective non-selectivity" where a small subset of receptors is activated to varying degrees by a specific ligand. Similarly, many psychoactive drugs are only effective because they target multiple GPCRs. For multi-component diseases such as drug abuse, the path to therapeutic intervention would benefit from being able to screen compounds for their effects across a wide range of GPCRs and select drugs with the proper activation profiles. Conversely, drugs that activate unintended targets can have disastrous effects. These compounds would also benefit from being able to assess their specificity across a wide range of toxicologically relevant GPCRs. In order to identify compounds that inhibit or activate multiple receptors of interest, a system is needed that profiles these responses simultaneously. Primity has developed a novel GPCR assay, applicable to greater than 90% of all GPCRs, that will be combined with Primity's cellular barcoding platform (CellCode) to enable the simultaneous screening 16 GPCR targets in a single assay well. In order to combine these techniques, the GPCR assay will first be optimized for performance in a high-throughput flow cytometry screening environment. Next, sixteen cell lines expressing different GPCRs will be generated and optimized for performance in the assay. To multiplex these targets, each cell line will be barcoded with a unique genetic identifier such that all sixteen cell lines can be mixed, frozen, thawed, and analyzed simultaneously. As a final validation, the mixture will be tested with specific agonists to ensure each cell line accurately reports the pharmacology of the test agonists (EC50) and antagonists (IC50) in combination and individually. The completion of these studies will provide a unique method of assaying receptor activation in a highly multiplexed system that dramatically reduces the time and costs of performing GPCR screens, yet ensures highly significant data generation. PUBLIC HEALTH RELEVANCE: The mechanisms and consequences of drug abuse cross multiple biological targets. The system described here is a method of identifying the effects of novel therapies across a broad range of targets to find unexpected activities of known drugs, psychoactive substances, and to define the components of herbal therapies, leading to the selection and development of better therapeutic compounds.

描述（由申请人提供）：第一阶段 SBIR 提案的主题是开发一个筛选系统，该系统可量化单个样本中 16 种不同 GPCR 中候选精神活性药物的影响。许多内源性 GPCR 配体靶向多种受体。特定的生物效应通常通过“选择性非选择性”来实现，其中一小部分受体被特定配体不同程度地激活。同样，许多精神活性药物之所以有效，是因为它们针对多个 GPCR。对于药物滥用等多成分疾病，治疗干预的途径将受益于能够在广泛的 GPCR 中筛选化合物的作用，并选择具有适当激活特性的药物。相反，激活非预期目标的药物可能会产生灾难性的影响。这些化合物也将受益于能够评估其在各种毒理学相关 GPCR 中的特异性。为了识别抑制或激活多个感兴趣受体的化合物，需要一个能够同时分析这些反应的系统。 Primity 开发了一种新型 GPCR 检测方法，适用于超过 90% 的 GPCR，该检测方法将与 Primity 的细胞条形码平台 (CellCode) 相结合，从而能够在单个检测孔中同时筛选 16 个 GPCR 靶标。为了结合这些技术，GPCR 检测将首先针对高通量流式细胞术筛选环境中的性能进行优化。接下来，将生成表达不同 GPCR 的 16 种细胞系，并针对检测中的性能进行优化。为了多重化这些靶标，每个细胞系都将带有唯一的遗传标识符，以便所有 16 个细胞系可以同时混合、冷冻、解冻和分析。作为最终验证，将使用特定激动剂对混合物进行测试，以确保每个细胞系准确报告测试激动剂 (EC50) 和拮抗剂 (IC50) 组合和单独的药理学。这些研究的完成将提供一种在高度多重系统中测定受体激活的独特方法，该方法可大大减少执行 GPCR 筛选的时间和成本，同时确保生成高度重要的数据。 公共卫生相关性：药物滥用的机制和后果涉及多个生物学目标。这里描述的系统是一种识别新疗法在广泛目标范围内的效果的方法，以发现已知药物、精神活性物质的意想不到的活性，并确定草药疗法的成分，从而选择和开发更好的治疗化合物。