New 5HT2AR Target Identification and Assay Development for Discovering Psychoplastogenic Compounds

具体目标： 新的5HT2AR/14-3-3亚型信号通路鉴定及检测进展 精神成形性化合物。 重要意义： 新出现的数据和有限的临床结果表明，迷幻药物有望在持久缓解 看似无法治愈的精神疾病和吸毒成瘾的人。神经影像证据 支持这样一个概念，即大脑在迷幻治疗后变得更灵活或更具可塑性，这 被认为是其假定的治疗作用的一个组成部分。然而，迷幻剂的副作用 可能是危险的甚至致命的。为了开发新的治疗方案，我们需要了解 迷幻潜在的行动机制。 许多致幻剂作用于G蛋白偶联受体5-HT2A型5-羟色胺受体(5HT2AR) (GPCR)，发挥它们的作用。G蛋白和β-arrestin信号通路不能区分治疗 以及迷幻剂的副作用。重要的是，5HT2AR拮抗剂酮丝氨酸，可以促进受体 内吞作用，并仍然从小鼠的抑郁相关行为中获得同等的缓解，这表明 人口贩运起着重要作用。 我们使用LinkLight技术研究了GPCR/14-3-3信号适配器的相互作用，发现 GPCR/14-3-3信号与受体转运密切相关。因此，研究5HT2AR/14-3-3信号 由迷幻剂介导的应该有助于理解有助于 迷幻和治疗的好处，并开发新的致精神病化合物。 我们建立了5HT2AR/14-3-3ε和5HT2AR/14-3-3γ联光分析方法。我们计划开发 额外的5HT2AR/14-3-3亚型分析包括14-3-3β和14-3-3ζ，因为GPCR可以区分 并暂时与14-3-3亚型接触。不同的14-3-3亚型具有不同的功能。 我们计划在5HT2AR/14-3-3亚型分析中分析一组迷幻药，看看我们是否可以连接 生理功能的信号通路。此外，我们希望进行一次试点筛查，以找到 化合物偏向于14-3-3信号通路，没有G蛋白信号。这种有偏见的化合物 可以引导我们更好地理解迷幻剂的潜在作用机制，并开发新的 精神成形性化合物。 实验设计： 任务1.开发额外5HT2AR/14-3-3β和5HT2AR/14-3-3ζ分析，5HT2AR/14-3-3 包括致幻剂和非致幻剂在内的一组致幻剂的同型异构体(ε，γ，β，&ζ)通路 致幻激动剂以及拮抗剂，并在不同和时间上表征 5HT2AR/14-3-3亚型信号。 任务2.通过进行以内部GPCR为重点的试点筛查，确定路径偏向的配体 5HT2AR/14-3-3亚型、G蛋白和β-arrestin途径HITS活性的文库和特征 化验。 下一阶段计划： 我们希望根据我们使用阿片受体/14-筛选的结果找到多途径偏向的配体。 3-3次化验(未发表结果)。将对这些多路径偏向配体进行初步评估 毒性和DMPK活性，并用于体内啮齿动物行为测试和神经成像。概念的证明 研究将使我们能够与潜在的合作者合作，加快药物开发。

Specific Aim: New 5HT2AR/14-3-3 isoform signal pathway identification and assay development for discovering psychoplastogenic compounds. Significance: Emerging data and limited clinical results show the promise of psychedelic drugs in the durable relief of people from seemingly untreatable psychiatric disorders and drug addictions. Neuroimaging evidence supports the concept that the brain after a psychedelic treatment becomes more flexible or `plastic', which is thought to be a component of its putative therapeutic effects. However, the side effects of psychedelics can be dangerous or even deadly. To develop new therapeutic options, we need to understand psychedelics underlying mechanisms of action. Many psychedelics act on the 5-HT2A serotonin receptor (5HT2AR), a G-protein Coupled Receptor (GPCR), to exert their effects. G-protein and β-arrestin signaling pathways cannot distinguish therapeutic and side effects of psychedelics. Importantly, the 5HT2AR antagonist ketanserin, can promote receptor endocytosis and still derive equivalent relief from depression-associated behaviors in mice, suggesting trafficking plays an important role. We studied GPCR/14-3-3 signal adaptor interactions using the LinkLight technology and found that GPCR/14-3-3 signals are closely related to receptor trafficking. Thus, studying 5HT2AR/14-3-3 signals mediated by psychedelics should shed light on understanding molecular mechanisms that contribute to psychedelic and therapeutic benefits, and develop new psychoplastogenic compounds. We have developed 5HT2AR/14-3-3ε and 5HT2AR/14-3-3γ LinkLight assays. We plan to develop additional 5HT2AR/14-3-3 isoform assays including 14-3-3β and 14-3-3ζ since a GPCR can differentially and temporally engage with 14-3-3 isoforms. Different 14-3-3 isoforms could display different functions. We plan to profile a panel of psychedelics in 5HT2AR/14-3-3 isoform assays and see if we can connect signaling pathways to physiological functions. In addition, we want to conduct a pilot screen to find compounds biased to the 14-3-3 signaling pathway without G-protein signals. Such biased compounds could lead us to better understand psychedelics' underlying mechanisms of action and develop new psychoplastogenic compounds. Experimental Design: Task 1. Develop additional 5HT2AR/14-3-3β and 5HT2AR/14-3-3ζ assays, profile 5HT2AR/14-3-3 isoform (ε, γ, β, & ζ) pathways with a panel of psychedelics including hallucinogenic and non- hallucinogenic agonists, as well as antagonists, and differentially and temporally characterize 5HT2AR/14-3-3 isoform signals. Task 2. Identify pathway-biased ligands by conducting a pilot screen with an in-house GPCR-focused library and characterize hits activities in 5HT2AR/14-3-3 isoforms, G-protein and β-arrestin pathway assays. Next Phase Plan: We expect to find multi-pathway biased ligands based on our results of screens using opioid receptor/14- 3-3 assays (unpublished results). These multi-pathway biased ligands will be evaluated for preliminary toxicity and DMPK activities and for in vivo rodent behavior tests and neuroimaging. The proof of concept studies will enable us to partner with potential collaborators to accelerate drug development.