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

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.