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

使用新开发的 MOR/14-3-3 生物测定法筛选调节 MOR 运输的分子。

• 批准号: 9900303
• 负责人: Haifeng Eishingdrelo
• 金额: $ 32.1万
• 依托单位: BIOINVENU CORPORATION
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900303
• 关键词: Address; Affect; Agonist; Animal Model; Animals; Arrestins; Biological Assay; Biotechnology; Cell membrane; Chemicals; Clinic; Clinical; Collaborations; Collection; Complex; Development; Disease; Dissociation; Dose; Drug Addiction; Drug Receptors; Endocytosis; Experimental Designs; FDA approved; Feasibility Studies; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Lead; Libraries; Life; Ligands; Link; Molecular Chaperones; Monitor; Morphine; Naloxone; Naltrexone; Neurobiology; Opiate Addiction; Opioid; Opioid Receptor; Opioid user; Overdose; Pain Threshold; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Play; Property; Proteins; Recruitment Activity; Recycling; Role; Savings; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Small Business Innovation Research Grant; Technology; Testing; Therapeutic; Therapeutic Agents; Time; Transportation; United States; Withdrawal; addiction; base; beta-arrestin; chronic pain; desensitization; drug development; high throughput screening; in vivo; mu opioid receptors; novel; novel strategies; novel therapeutics; opiate tolerance; opioid epidemic; opioid mortality; opioid overdose; opioid use disorder; pain model; receptor; response; screening; small molecule; small molecule libraries; therapeutic development; trafficking

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.

具体目标： 使用试点 HTS 验证 MOR/14-3-3 LinkLight，以识别分子测定调节 铁道部贩运。 意义： 美国当前的阿片类药物使用障碍 (OUD) 和阿片类药物过量死亡危机 各国迫切需要更安全、更好的治疗方法来开发更好、更安全的治疗方法。 治疗 OUD 的药物，首先也是最重要的是进行新的生物测定， 与当前的方法不同，并且与潜在的生理相关 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 确定 OUD 药物开发的新治疗线索。 我们开发了 MOR/14-3-3 LinkLight 检测，我们发现 14-3-3 蛋白参与其中。 MOR/14-3-3 关联信号可以由 MOR 促进。 拮抗剂纳洛酮（救命过量药物）和纳曲酮（抗成瘾药物）。 内啡肽可以破坏 MOR/14-3-3 相互作用信号，同时促进 MOR/β-抑制蛋白相互作用。 MOR1 内吞和运输在阿片类药物耐受性和阿片类药物耐受性的发展中发挥着关键作用 因此，MOR/14-3-3 LinkLight 生物测定代表了前所未有的技术。 筛选和鉴定治疗 OUD 的新治疗剂的机会。 实验设计： 任务 1. 对调节 MOR1/14-3-3 相互作用信号的小分子进行试点筛选。 我们计划筛选大约10,000个小分子库，包括批准的药物 化合物库、临床化合物库、GPCR化合物库和生物活性化合物库。 任务 2. 表征从 G 蛋白和 β-arrestin 筛选中识别出的阳性命中 MOR/14-3-3 是 MOR/14-3-3 LinkLight 鉴定的新途径测定。 G 蛋白和 β-arrestin 信号通路的检测可能具有不同的特性。 我们开发了基于 G 蛋白信号分析的激动剂和拮抗剂分类。 MOR/β-arrestin LinkLight 测定和该测定将用于评估 β-arrestin 的命中 基于三种途径检测，我们期望发现多途径偏倚。 这些多途径偏向的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基于机制的新颖方法的可行性 一旦我们证明了可行性，就将进行第二阶段的筛选和鉴定。 计划包括筛选更大的化合物集合、评估 DMPK 的阳性结果 动物研究，并评估动物模型对疼痛、耐受性、戒断、 和成瘾，并且在与 NCATS 的潜在合作中，我们将提出合作。 与适当的制药公司合作，尽可能加速开发。 概括： 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。 MOR/14-3-3 LinkLight 分析发现 14-3-3 蛋白参与 MOR 运输。 MOR/14-3-3 测定可用于筛选调节 MOR 运输的分子。 从拟议的筛选中识别出的潜在命中可能具有治疗药物的潜力 发展。