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

今天，数百万美国人患有阿片类药物使用障碍（OUD）。数百万人滥用 阿片类药物，危机继续增长。该提案的目的是加快 非成瘾性镇痛剂的发现，为药物发现团队提供更简单， 更稳健、更定量的激动剂偏倚测定。在紧急情况下， 我们正在寻求快速通道支持，以创建新的检测和分析工具， 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， 激动剂偏向于与Gi和β-抑制蛋白信号传导偶联的特定受体 途径，并创建新的工具，以改善结构/活性关系的分析。 有很好的理由寻找在文献中鉴定的受体的偏向性激动剂。 NIDA“十大”药物开发重点名单。偏性激动剂可以激活 有益的信号通路，同时避免那些导致不良影响的信号通路。找到 这些偏向性激动剂是困难的：目前用于检测偏向性的测定法， 并且已经过验证，但是存在限制动物模型的可转化性的缺点 和临床研究。这些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。后者的结果在时间依赖性的偏置 使体内疗效预测和SAR复杂化的测量 通过添加额外的变量。 我们的新测定将同时测量在细胞中Gi和β-arrestin信号传导的动力学。 活细胞这个项目将涉及创建新的工具，以及重新利用的 我们已经开发出了研究非OUD药物靶点的方法。将对测定进行优化 用于标准荧光板读取器，数据分析工具箱将 开发用于简化基于动力学测量的激动剂偏差的定量。 第一阶段将完成对NOP阿片受体的初步验证研究，目标是 证明检测可靠性和灵敏度里程碑。第二阶段将优化 测定D3多巴胺、CB 1大麻素和OPRM 1阿片受体，并开发 用于部署在标准酶标仪和软件包上的分析工具箱 通常用于药物发现。在第二阶段的下半部分， 协议将分发给正在开发治疗OUD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.