Multiplexed Measurement of Psychoactive Drug Selectivity Profiles

描述（由申请人提供）：本I期SBIR提案的主题是开发一种筛选系统，该系统可在单个样本中对候选精神活性药物在16种不同GPCR中的作用进行量化。许多内源性GPCR配体靶向多种受体。特定的生物学效应通常通过“选择性非选择性”来实现，其中受体的小子集被特定配体激活到不同程度。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。对于药物滥用等多组分疾病，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征的药物。相反，激活非预期目标的药物可能会产生灾难性的影响。这些化合物还将受益于能够评估其在广泛的毒理学相关GPCR中的特异性。为了鉴定抑制或激活多种感兴趣受体的化合物，需要同时描绘这些反应的系统。Priestival开发了一种新型GPCR检测方法，适用于90%以上的GPCR，该方法将与Priestival的细胞条形码平台（CellCode）相结合，以便在单个检测孔中同时筛选16个GPCR靶标。为了将这些技术联合收割机组合，首先将针对高通量流式细胞术筛选环境中的性能对GPCR测定进行优化。接下来，将产生表达不同GPCR的16个细胞系，并针对测定中的性能进行优化。为了多重化这些靶标，将用独特的遗传标识符对每个细胞系进行条形码化，使得所有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检测，以确保各细胞系准确报告试验激动剂（EC50）和拮抗剂（IC50）的联合和单独药理学。这些研究的完成将提供一种独特的方法，在一个高度多路复用的系统中测定受体激活，大大减少了进行GPCR筛选的时间和成本，但确保高度重要的数据生成。 公共卫生相关性：药物滥用的机制和后果跨越多个生物靶点。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、精神活性物质的意外活性，并定义草药疗法的组分，从而选择和开发更好的治疗化合物。

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.