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Author(s): Peiyu Xu [sup.1] [sup.2] [sup.3] , Sijie Huang [sup.2] [sup.3] [sup.4] , Huibing Zhang [sup.1] [sup.5] [sup.6] [sup.7] , Chunyou Mao [sup.1] [sup.5] [sup.6] [sup.7] , X. Edward Zhou [...], Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter.sup.1,2 that activates the largest subtype family of G-protein-coupled receptors.sup.3. Drugs that target 5-HT.sub.1A, 5-HT.sub.1D, 5-HT.sub.1E and other 5-HT receptors are used to treat numerous disorders.sup.4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT.sub.1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT.sub.1D bound to 5-HT; and 5-HT.sub.1E in complex with a 5-HT.sub.1E- and 5-HT.sub.1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT.sub.1A interface, and is able to increase 5-HT.sub.1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules--particularly in the case of 5-HT.sub.1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT.sub.1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors. Cryo-electron microscopy structures of three different serotonin receptors in complex with serotonin and other agonists provide insights into the role of lipids in regulating these receptors and the structural basis of ligand recognition.

The serotonin 7 receptor (5-HT 7 R) regulates various processes in the central nervous system, including mood, learning, and circadian rhythm control, among others. Receptor activation can lead to activation of the Gα s protein and a subsequent increase of intracellular cyclic adenosine monophosphate (cAMP). Receptor interaction with inverse agonists results in a decrease of basal cAMP levels and therefore a downstream effect of reduced neuronal excitability and neurotransmission. Recently, pellotine ( 1a ), a Lophophora alkaloid, was unexpectedly shown to be an inverse agonist of the 5-HT 7 R. Therefore, we evaluated close analogs of compound 1a , both naturally occurring and synthetic analogs, as inverse agonists of the 5-HT 7 R. Functional evaluation in a GloSensor cAMP assay revealed a preference for an 8-hydroxy-6,7-dimethoxy substitution pattern over 6,7,8-trimethoxy analogs or 8-hydroxy-6,7-methylenedioxy analogs. This was supported by molecular dynamics simulations, where the 8-hydroxy substitution allowed more robust interaction with the 5-HT 7 R, which correlated with inverse agonism efficacy. Additionally, N -methylation (as in 1a ) improved the potency of the evaluated analogs. In this series, the most potent inverse agonist was anhalidine ( 1b ) (EC 50 = 219 nM, E max ), and showed a 2-fold higher functional potency. Altogether, these results provide key insights for the further development of potent low molecular weight inverse agonists of the 5-HT 1a ), and showed a 2-fold higher functional potency. Altogether, these results provide key insights for the further development of potent low molecular weight inverse agonists of the 5-HT 7 R.

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT 2A ) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT 2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N -benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGα q to the 5-HT 2A , allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC 50 and E values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGα max - than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT q may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).

The serotonin 2A receptor (5-HT 2A R) is the mediator of the psychedelic effects of serotonergic psychedelics, which have shown promising results in clinical studies for several neuropsychiatric indications. The 5-HT 2A R is able to signal through the Gα q and β-arrestin effector proteins, but it is currently not known how the different signaling pathways contribute to the therapeutic effects mediated by serotonergic psychedelics. In the present work, we have evaluated the subtype-selective 5-HT 2A R agonist 25CN-NBOH and a series of close analogues for biased signaling at this receptor. These ligands were designed to evaluate the role of interactions with Ser159 3×36 . The lack of interaction between this hydroxyl moiety and Ser159 3×36 resulted in detrimental effects on potency and efficacy in both βarr2 and miniGα q recruitment assays. Remarkably, Gα q -mediated signaling was considerably more affected. This led to the development of the first efficacious βarr2-biased 5-HT 2A R agonists 4a-b and 6e-f , βarr2 preferring, relative to lysergic acid diethylamide (LSD).