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

具体目标： 新的5 HT 2AR/14-3-3同种型信号通路鉴定和测定开发用于发现 致精神质化合物 重要性： 新出现的数据和有限的临床结果表明，迷幻药有望持久缓解 人们从看似无法治愈的精神疾病和药物成瘾。神经影像学证据 支持这样的概念，即大脑在迷幻治疗后变得更加灵活或“可塑性”， 被认为是其推定的治疗效果的组成部分。然而迷幻药的副作用 可能很危险甚至致命为了开发新的治疗方案，我们需要了解 致幻剂的潜在作用机制。 许多致幻剂作用于5-HT 2A血清素受体（5 HT 2AR），一种G蛋白偶联受体 （GPCR），以发挥其作用。G蛋白和β-抑制蛋白信号通路不能区分治疗性 以及迷幻药的副作用重要的是，5 HT 2AR拮抗剂酮色林可以促进受体的表达。 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 贩运人口起着重要作用。 我们使用LinkLight技术研究了GPCR/14-3-3信号衔接子相互作用，发现 GPCR/14-3-3信号与受体转运密切相关。因此，研究5 HT 2AR/14-3-3信号， 通过迷幻剂介导的，应该有助于理解分子机制， 迷幻和治疗的好处，并开发新的精神可塑性化合物。 我们已经开发了5 HT 2AR/14-3-3ε和5 HT 2AR/14-3-3γ LinkLight测定。我们计划开发 由于GPCR可以差异化地检测14-3-3β和14 - 3 - 3 β，因此额外的5 HT 2AR/14- 3 -3亚型测定包括14-3-3β和14-3-3 β。 暂时与14-3-3同种型结合不同的14-3-3异构体具有不同的功能。 我们计划在5 HT 2AR/14-3-3同种型检测中分析一组致幻剂，看看我们是否能将其与 生理功能的信号通路。另外，我们要进行试点筛选， 化合物偏向于14-3-3信号通路而没有G蛋白信号。这种有偏见的化合物 可以让我们更好地理解迷幻药的潜在作用机制， 致精神质化合物 实验设计： 任务1.开发额外的5 HT 2AR/14-3-3β和5 HT 2AR/14-3-3 β测定，分析5 HT 2AR/14-3-3 同种型（ε，γ，β，& ε）通路与一组致幻剂，包括致幻剂和非致幻剂 致幻激动剂，以及拮抗剂，并差异和时间表征 5 HT 2AR/14-3-3亚型信号。 任务2.通过使用内部GPCR聚焦的中试筛选， 文库和表征5 HT 2AR/14-3-3同种型、G蛋白和β-抑制蛋白途径中的命中活性 测定。 下一阶段计划： 我们期望基于我们使用阿片受体/14-受体筛选的结果找到多途径偏向的配体。 3-3分析（未发表的结果）。将对这些多途径偏向配体进行初步评估， 毒性和DMPK活性以及体内啮齿动物行为测试和神经成像。概念验证 研究将使我们能够与潜在的合作者合作，加速药物开发。

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.