Screen molecules modulating MOR trafficking with a newly developed MOR/14-3-3 bioassay.

具体目标： 使用中试HTS验证莫尔/14 - 3 - 3 LinkLight试验，以鉴定调节分子 莫尔走私。 重要性： 目前美国阿片类药物使用障碍（OUD）和阿片类药物过量死亡的危机 各国迫切需要更安全和更好的治疗方法。开发更好更安全的治疗药物 治疗OUD的药物，首先是要有新的生物测定， 与目前的方法不同，并且与潜在的 阿片类药物成瘾和慢性疼痛的神经生物学，并使用生物测定来筛选和 为OUD药物开发确定新的治疗线索。 我们开发了莫尔/14 - 3 - 3 LinkLight检测法。我们发现14 - 3 - 3蛋白参与了 气相化学还原剂的贩运和回收。莫尔/14 - 3 - 3关联信号可被莫尔促进 拮抗剂纳洛酮（救命过量药物）和纳洛酮（抗成瘾药物）。Mor 内吗啡肽可以破坏莫尔/14 - 3 - 3相互作用信号，同时促进 莫尔/β-抑制蛋白相互作用。 MOR1的内吞和运输在阿片耐受的发展中起关键作用， 依赖因此，莫尔/14 - 3 - 3 LinkLight生物测定法代表了前所未有的 筛选和鉴定治疗OUD的新治疗剂的机会。 实验设计： 任务1.进行调节MOR1/14 - 3 - 3相互作用信号的小分子的中试筛选。 我们计划筛选大约10，000个小分子文库，包括已批准的药物 文库、临床化合物文库、GPCR化合物文库和生物活性化合物文库。 任务2.表征从G蛋白和β-抑制蛋白筛选中鉴定的阳性命中 途径。莫尔/14 - 3 - 3是一种新的途径试验。由莫尔/14 - 3 - 3 LinkLight识别的命中 测定可能对G蛋白和β-抑制蛋白信号传导途径具有不同的性质。我们将 基于G蛋白信号传导测定对激动剂和拮抗剂进行分类。我们已经开发 莫尔/β-抑制蛋白LinkLight检测试剂盒，该检测试剂盒将用于评价β-抑制蛋白的命中率 途径活性。基于这三个途径的分析，我们期望发现多途径偏倚 配体。这些多途径偏向配体将增强我们对神经生物学的理解， 阿片类药物成瘾和慢性疼痛，以及引导我们开发更好，更安全的药物。 下一阶段计划： 第一阶段项目将测试这种新的基于机制的方法的可行性， 筛选和鉴定OUD的新分子。一旦我们证明了可行性， 该计划包括筛选更大的化合物集合，评估DMPK的阳性命中 在动物中的研究，并评估疼痛，耐受性，戒断， 和成瘾，并在潜在的合作与NCATS将提出。我们将与 与适当的制药公司合作，在可能的情况下加快开发。 摘要： 越来越多的证据表明，阿片受体脱敏和贩运 机制与耐受性和成瘾性密切相关。我们已经开发出一种新颖 莫尔/14 - 3 - 3 LinkLight测定。我们发现14 - 3 - 3蛋白参与莫尔运输。因此 莫尔/14 - 3 - 3测定可用于筛选调节莫尔运输的分子。的 从所提出的筛选中鉴定的潜在命中物可能具有治疗剂的潜力， 发展

Specific Aim: Validate the MOR/14-3-3 LinkLight assay with a pilot HTS to identify molecules modulating MOR trafficking. Significance: The current crisis of opioid use disorders (OUDs) and opioid overdose deaths in the United States urgently demands safer and better therapeutics. To develop better and safer therapeutic agents for the treatment of OUDs, first and foremost is to have new bioassays which are different from the current approaches and are physiologically relevant to the underlying neurobiology of opioid addiction and chronic pain, and to use the bioassays to screen and identify new therapeutic leads for OUD drug development. We have developed MOR/14-3-3 LinkLight assay. We found that 14-3-3 proteins participate in GPCR trafficking and recycling. MOR/14-3-3 association signals can be promoted by MOR antagonists naloxone (live-saving overdose drug) and naltrexone (anti-addiction drug). MOR endomorphins can disrupt MOR/14-3-3 interaction signals, while at the same time promote MOR/β-arrestin interaction. MOR1 endocytosis and trafficking play a critical role in the development of opioid tolerance and dependence. Thus, the MOR/14-3-3 LinkLight bioassay represents an unprecedented opportunity for screen and identification of new therapeutic agents for the treatment of OUDs. Experimental Design: Task 1. Conduct a pilot screen of small molecules modulating MOR1/14-3-3 interaction signals. We plan to screen approximate total 10,000 small molecule libraries including approved drug library, clinic compound library, GPCR compound library, and bioactive compound library. Task 2. Characterize positive hits identified from the screen for G-protein and β-arrestin pathways. MOR/14-3-3 is a new pathway assay. The hits identified by MOR/14-3-3 LinkLight assay may have different properties for G-protein and β-arrestin signaling pathways. We will classify agonists and antagonists based on G-protein signaling assay. We have developed MOR/β-arrestin LinkLight assay and the assay will be used to evaluate the hits for β-arrestin pathway activity. Based on the three pathway assays, we expect to find multi-pathway biased ligands. These multi-pathway biased ligands will enhance our understanding neurobiology of opioid addiction and chronic pain, as well as lead us to develop better and safer drugs. Next Phase Plan: The phase I project will test the feasibility of this novel mechanism-based approach for screening and identifying new molecules for OUDs. Once we demonstrate feasibility, a phase II plan that includes screening larger compound collections, evaluating positive hits for DMPK studies in animals, and evaluating positive hits for animal models of pain, tolerance, withdrawal, and addiction, and in potential collaboration with NCATS will be proposed. We will partner the project with appropriate pharmaceutical companies to accelerate development when possible. Summary: Accumulating evidence suggests that opioid receptor desensitization and trafficking mechanisms are intimately connected to tolerance and addiction. We have developed a novel MOR/14-3-3 LinkLight assay. We found 14-3-3 proteins involved in MOR trafficking. Thus, the MOR/14-3-3 assay can be utilized for screening molecules modulating MOR trafficking. The potential hits identified from the proposed screen could have potentials for therapeutic agent development.