An optimized screening platform for identifying and quantifying biased agonists as drugs for the treatment of Opioid Use Disorder

今天数以百万计的美国人患有阿片类药物使用障碍(OUD)。又有数百万人被滥用 阿片类药物，而且危机继续加剧。这项提议的目标是加快 通过为药物发现团队提供更简单、 更可靠、更定量的激动剂偏倚分析。在紧迫性的推动下 问题我们正在寻求Fast Track支持，以创建新的分析和分析工具 OUD研究中的药物发现。我们的目标是优化和测试新的检测方法 与Gi和β-arrestin信号偶联的特定受体上的激动剂偏向 途径，并创造新的工具来改进结构/活性关系的分析。 有充分的理由寻找有偏见的激动剂，以识别在 NIDA列出了药物开发的十大优先事项。有偏向的激动剂可以激活 有益的信号通路，而避免那些引起不利影响的信号通路。查找 这些有偏见的激动剂是困难的：目前检测偏见的检测方法，虽然已经确定 并经过验证，但存在限制动物模型可译性的缺陷 和临床研究。这些条件包括完全不同的实验条件 测量被比较的不同信号通路和不同的时间进程 被测量的反应。后者导致偏差的时间依赖性 使体内疗效预测复杂化并使SAR复杂化的测量 表中添加额外的变量。 我们的新方法将同时测量Gi和β-arrestin信号的动力学。 活细胞。该项目将包括创建新工具以及重新调整工具的用途 我们已经发展到研究非OUD药物靶点。化验结果将得到优化 用于标准荧光平板读取器，并将有一个数据分析工具箱 开发用于简化基于动力学测量的激动剂偏倚的量化。 第一阶段将完成对NOP阿片受体的初步验证研究，目标是 展示了检测的可靠性和灵敏度里程碑。第二阶段将优化 D3多巴胺、CB1类大麻素和OPRM1阿片受体的检测及进展 用于在标准板材阅读器和软件包上部署的分析工具箱 通常用于药物发现。在第二阶段的后半部分，详细的分析 协议将准备好分发给正在为OUD开发新药的研究人员。

Millions of Americans today have an opioid use disorder (OUD). Millions more misuse opioids, and the crisis continues to grow. The goal of this proposal is to speed the discovery of non-addictive analgesics by providing drug discovery teams with simpler, more robust, more quantitative, assays for agonist bias. Driven by the urgency of the problem we are seeking Fast Track support to create new assay and analytic tools for drug discovery in OUD research. Our goal is to optimize and test new assays for agonist bias at particular receptors that couple to both the Gi and β-arrestin signaling pathway, and create new tools to improve the analysis of structure/activity relationships. There are good reasons to search for biased agonists to the receptors identified in the NIDA “top ten” list of medication development priorities. Biased agonists could activate beneficial signaling pathways while avoiding those that cause adverse effects. Finding these biased agonists is difficult: current assays for detecting bias, while established and validated, suffer from drawbacks that are limiting translatability to animal models and clinical studies. These include entirely different sets of experimental conditions for measuring the different signaling pathways being compared and different time courses of the response being measured. The latter results in time-dependence of the bias measurement which complicates predictions of in vivo efficacy and complicates SAR tables by adding extra variables. Our new assay will simultaneously measure the kinetics of Gi and β-arrestin signaling in living cells. This project will involved creating new tools as well as re-purposing ones we have already developed to study non-OUD drug targets. The assay will be optimized for use on standard fluorescence plate readers, and a data analysis toolbox will be developed to simplify quantification of agonist bias based on kinetic measurements. Phase I will complete the initial validation studies on the NOP opioid receptor, with goal of demonstrating assay reliability and sensitivity milestones. Phase II will optimize the assay for D3 dopamine, CB1 cannabinoid and OPRM1 opioid receptors and develop the analysis toolbox for deployment on standard plate readers and software packages commonly used in drug discovery. In the second half of Phase II, assays with detailed protocols will be ready distribute to researchers who are developing new drugs for OUD.