Identification of 5-HT 2A Receptor Signaling Pathways Responsible for Psychedelic Potential

Wallach and colleagues frame the study in the context of renewed interest in serotonergic psychedelics for rapid and sustained therapeutic effects, alongside enduring uncertainty about which 5-HT2A receptor (5-HT2AR) signalling pathways mediate their hallucinogenic versus putative therapeutic actions. Classical psychedelics from diverse chemical scaffolds all activate 5-HT2AR, and human and preclinical work (including blockade of subjective effects by the antagonist ketanserin and use of the mouse head-twitch response (HTR) as a predictive behavioural assay) point to 5-HT2AR as central to psychedelic effects. However, prototypical psychedelics engage both Gq/11 proteins and β-arrestin2 via 5-HT2AR, leaving the relative contribution of these transducers unclear and complicating rational design of new ligands that might separate therapeutic benefit from hallucinogenic liability. To address this gap, the investigators used structure-guided medicinal chemistry to create a series of 5-HT2AR-selective ligands with graded Gq efficacies, including β-arrestin2-biased agonists, and then used a battery of in vitro signalling assays, structural modelling and in vivo behavioural tests (chiefly the HTR in male mice) to probe which 5-HT2AR-coupled pathways predict psychedelic potential. The study aims to identify whether Gq or β-arrestin2 efficacy at 5-HT2AR determines psychedelic-like effects and to define structural features that enable tuning of 5-HT2AR signalling for drug development.

The experimental strategy combined medicinal chemistry, in vitro pharmacology, structural modelling and mouse behavioural pharmacology. Chemists synthesised an extended series of N-benzyl-phenethylamine derivatives (the 25N series) and related analogues using standard reductive amination and characterised products by HPLC, HRMS, elemental analysis and NMR; full synthetic details and analytical data are reported in the Supplementary document. Signalling was quantified primarily with BRET-based assays: Gq activation was measured using a Gαq–Gγ1 dissociation BRET2 configuration, and β-arrestin2 recruitment was measured using a RLuc8–Venus BRET1 assay in HEK293T cells. Assays were run at multiple time points to account for ligand kinetics. Complementary functional readouts included calcium-flux assays in inducible Flp-In 293 cells to assess Gq/11-mediated responses, competitive radioligand binding (performed by the NIMH PDSP) for affinity across 5-HT subtypes and off-targets, and NanoBit HiBiT surface-expression/internalisation assays to measure ligand-induced receptor endocytosis. Structure–function work comprised induced-fit docking into the Gq-bound 5-HT2AR cryo-EM structure and molecular dynamics (MD) simulations in a lipid bilayer to examine ligand interactions and conformational consequences, with particular attention to residue W336 (W6.48). Site-directed mutagenesis (e.g. W336Y, W336L) was used to test hypotheses arising from modelling. Behavioural pharmacology used male C57BL/6J mice. The head-twitch response (HTR) was measured with a magnet/coil detection system in surgically magnet-implanted mice; dose–response curves and ED50s were estimated by nonlinear regression. Locomotor hyperactivity induced by phencyclidine (PCP) was assessed in the behavioural pattern monitor (BPM) as distance travelled. Tolerance/tachyphylaxis experiments involved once-daily dosing for five days followed by DOI challenge. To interrogate causality for Gq-PLC signalling, the selective Gq/11 inhibitor YM-254890 was administered intracerebrally (ICV) and the PLC inhibitor edelfosine was given systemically before psychedelic challenge. Statistical analyses included one-way ANOVAs (or Welch ANOVA), post hoc tests, and correlation analyses (Spearman or Pearson as reported) comparing in vitro efficacy measures (Emax relative to 5-HT) with HTR magnitude.

Initial BRET experiments showed that classical psychedelics (e.g. psilocin, DMT, 2C-I, LSD, psilocybin metabolites) engage both 5-HT2AR-Gq and 5-HT2AR-β-arrestin2 with largely balanced efficacy when compared at equivalent time points; some ligands exhibited time-dependent increases (in some cases exceeding 5-HT activity at long times), but no strong bias for one transducer was observed across the tested psychedelics. From the 25N phenethylamine scaffold, the team developed multiple N-benzyl analogues and identified compounds with improved 5-HT2AR selectivity. Notably, 25N-NBI (compound 10) showed 23-fold selectivity for 5-HT2AR over 5-HT2CR and induced the HTR in mice (ED50 = 10.9 μmol/kg), confirming that 5-HT2AR-selective agonists can have psychedelic potential. Structure–activity analyses suggested that ring electrostatics and steric bulk at N-benzyl positions modulate affinity, selectivity and signalling bias; docking and mutagenesis implicated aromatic anchor residues (F339, F340) in affinity. Replacing the N-benzyl ring with bulky bi-aryl groups (N-naphthyl or N-biphenyl) produced compounds (25N-N1-Nap (16) and 25N-NBPh (17)) with substantially reduced Gq efficacy but preserved β-arrestin2 recruitment, i.e. β-arrestin-biased agonism at 5-HT2AR. MD simulations showed that these bulky ligands push W336 (W6.48) into an alternative rotamer conformation; a W336Y mutation restored Gq efficacy for biased ligands, supporting a steric toggle-switch mechanism for bias. Behaviourally, five distinct β-arrestin2-biased 5-HT2AR agonists failed to induce the HTR in male mice at doses that blocked the HTR induced by the balanced agonist DOI, and they antagonised 5-HT2AR-Gq signalling in vitro with potencies comparable to the selective antagonist M100907. Cross-scaffold N-naphthyl and N-biphenyl derivatives similarly showed weak Gq efficacy, strong β-arrestin2 recruitment, failure to produce HTR, and blockade of DOI-induced HTR, indicating the biased, non-psychedelic profile generalises beyond the initial 25N series. Correlation analyses across the 25N series (n=14) revealed a strong positive relationship between HTR magnitude (max counts/minute) and 5-HT2AR-Gq efficacy (%5-HT EMAX; Spearman R S = 0.8242, p = 0.0005), whereas there was no correlation with β-arrestin2 recruitment (R S = -0.01538, p = 0.9638). The relationship was nonlinear: none of the 25N derivatives with Gq EMAX <70% induced HTR. Extending this analysis to 24 phenethylamine psychedelics yielded a similar finding (R S = 0.7339, p<0.0001 for Gq efficacy vs HTR; no significant correlation with β-arrestin2). Predictive syntheses based on a 70% Gq efficacy threshold were confirmed experimentally: highly efficacious Gq agonists (Emax ~96–100%) induced robust HTRs, whereas compounds with Emax <70% did not. Pharmacological inhibition of Gq-PLC signalling provided convergent causal evidence: intracranial pretreatment with YM-254890 blocked DOI-induced HTR, and systemic edelfosine blocked the HTR elicited by DOI and 25N-NBOMe. Comparison of classical psychedelics and putative non-psychedelic 5-HT2AR agonists showed that LSD, psilocin, 5-MeO-DMT and DET had Gq EMAX between 74.1–98.8% and induced HTR, while lisuride, 2-Br-LSD, 6-F-DET and 6-MeO-DMT had Gq EMAX <70% and did not induce HTR. For these eight compounds, HTR magnitude correlated with Gq EMAX (R = 0.8948, p = 0.0027). Cellular internalisation assays (NanoBit) demonstrated that β-arrestin-biased agonists (25N-N1-Nap and 25N-NBPh) induced robust β-arrestin2-dependent 5-HT2AR internalisation, unlike the inverse agonist pimavanserin. In vivo, once-daily administration of 25N-N1-Nap or DOI for five days reduced the HTR to a DOI challenge 24 h later, indicating tachyphylaxis; repeated pimavanserin did not produce this effect. Finally, 25N-N1-Nap attenuated PCP-induced locomotor hyperactivity in mice at doses that did not alter baseline activity, an effect similar to M100907, consistent with an antipsychotic-like profile.

Wallach and colleagues interpret their data as identifying 5-HT2AR-Gq efficacy, rather than β-arrestin2 recruitment, as the key predictor of psychedelic-like activity measured by the mouse HTR. They emphasise a putative efficacy threshold—approximately 70% of 5-HT EMAX in their assays—below which compounds do not induce head twitches, offering a mechanistic explanation for why certain 5-HT2AR agonists (for example lisuride and 2-Br-LSD) lack psychedelic effects despite receptor engagement. The investigators note that classical psychedelics activate both Gq and β-arrestin2, but β-arrestin-biased agonists were devoid of acute psychedelic-like behavioural effects while still inducing receptor internalisation and tachyphylaxis, and producing antagonist-like behavioural outcomes such as blockade of PCP-induced hyperactivity. Structurally, the authors argue that steric interactions with W336 (W6.48) in the orthosteric/extended binding pocket can shift 5-HT2AR conformational equilibria to favour arrestin-preferring states, and that docking, MD and mutagenesis data support this toggle-switch mechanism. They propose that this understanding enables rational design of 5-HT2AR ligands with tailored profiles—partial Gq agonists that remain below the psychedelic threshold yet may retain therapeutic neurophysiological actions (for example, neuroplasticity), and β-arrestin-biased ligands that mimic aspects of antagonists with potential antipsychotic-like utility. The authors acknowledge limitations and uncertainties reported in the paper. Previous studies in constitutive knockout mice yielded inconsistent results for Gq and β-arrestin2 roles in the HTR, and adaptive changes in global KOs complicate interpretation. They also note potential species differences between rodent and human 5-HT2AR pharmacology and call for further structural studies to map additional conformational determinants of Gq versus arrestin coupling. Finally, the paper highlights the need to test partial Gq agonists and β-arrestin-biased ligands in disease-relevant preclinical models to determine whether therapeutic benefits can be dissociated from psychedelic effects; these are presented as future directions rather than established clinical claims.