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

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, and 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.