PROJECT SUMMARY The rise of interest in psychedelic medicine calls for further exploration of the mechanisms underlying the therapeutic nature of these drugs. Serotonin receptors are well-established regulators of behavioral and cognitive effects. In particular, the serotonin receptor 5-HT1A is a primary target of antidepressant, anxiolytic, and antipsychotic medications. 5-HT1A is also a high-affinity target of multiple psychedelic drugs, but its role in the physiological effects of psychedelics has been overlooked as the serotonin receptor 5-HT2A is suggested to be the primary mediator of hallucinogenic effects. However, select psychedelics have equivalent or greater affinity for 5-HT1A over 5-HT2A. This project seeks to determine the unique molecular details of psychedelic binding and activity at 5-HT1A with structural and functional studies. Structures of a 5-HT1A-G-protein complex bound to psychedelic and therapeutic drugs will be solved via cryo-electron microscopy, revealing ligand and transducer binding interactions at near-atomic resolution. This project will also address the lack of receptor- level information on psychedelic activity with a series of in vitro functional studies. Distinct signaling pathways have been associated with therapeutic or undesirable effects at other G-protein coupled receptors. As such, the activity of existing psychedelics at 5-HT1A and 5-HT2A will be characterized across two major signaling pathways—G-protein and b-arrestin. Additionally, the psychedelic 5-methoxy-dimethyltryptamine (5-MeO- DMT) is reportedly selective for 5-HT1A over 5-HT2A. This scaffold will be exploited. A series of 5-MeO-DMT derivatives will be synthesized and tested to identify the pharmacophores conferring selectivity for 5-HT1A over 5-HT2A. This experiment will generate selective compounds useful for delineating the role of 5-HT1A in vivo. In a parallel set of experiments, 5-HT1A will be mutagenized to identify individual residues that play a key role in 5-HT1A activity and signaling. These experiments will confirm structural observations and identify important residues outside of 5-HT1A’s ligand binding pocket. The complementary results of structural and functional experiments will serve as an invaluable resource for future in vivo and drug-design efforts. In summary, the proposed studies will reveal the structural and chemical determinants of psychedelic activity at 5-HT1A and generate novel psychedelic compounds with defined activity, which can be used in future studies to delineate the precise role of 5-HT1A in psychedelic physiology.