Psychedelics exhibit similar Gq and β-arrestin2 activity at 5-HT2AR
To investigate the 5-HT2AR signaling profiles of classical psychedelics, we used bioluminescence resonance energy transfer approaches (BRET, Fig. 1A, B, Supplementary Table 1), which provide a proximity measure of intracellular transducer engagement. BRET is used extensively to quantify GPCR-biased agonism and has the advantage of not being susceptible to second messenger amplification or receptor reserve issues obfuscating GPCR signaling preference determinations20. BRET has been used to measure 5-HT2AR-β-arrestin2 and 5-HT2AR-Gq activity directly11, and to confirm 5-HT2AR G protein coupling preferences18. In our assay platform, 5-HT2AR strongly couples to Gq/11 and β-arrestin2 over all other G protein subtypes and β-arrestin1 (Fig. 1C).
Fig. 1: Psychedelics exhibit similar Gq and β-arrestin2 activity at 5-HT2AR.
Schematic of 5-HT2A receptor G protein dissociation (A) and β-arrestin recruitment (B) to determine transducer preferences. (C) Determination of 5-HT2A receptor G protein-wide and β-arrestin1/2 transducer preferences as estimated by magnitude net BRET for each transducer as stimulated by 5-HT. Data represent the mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubations. D–K Comparison of 5-HT2A receptor Gq dissociation (red) and β-arrestin2 recruitment (blue) for 5-HT (D) and several classes of psychedelics: (E) Psilocin, (F) DMT, (G) 5-MeO-DMT, (H) 2C-I, (I) DOI, (J) 25I-NBOMe, K LSD. Data represent the mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubations. L Heat map of Gq dissociation and β-arrestin2 recruitment kinetics displayed as log (EMAX/EC50) for the psychedelics tested. Data represent the mean and SEM from three independent experiments, which were performed at 37 °C at the indicated compound incubation time points. Source data are provided as a Source Data file.
Next, we tested classical psychedelics from multiple chemical classes and examined their effects on Gq dissociation and β-arrestin2 recruitment (Fig. 1D-K). Kinetic issues can confound studies of ligand-directed bias21, and slow ligand kinetics can delay full receptor occupancy, as observed for LSD at 5-HT2AR19. Therefore, we thoroughly assessed Gq and β-arrestin2 activities at various time points at 37 °C to ensure full receptor occupancy and confirm that transducer preferences do not substantially change when compared at the same time point. Our results show that psychedelics exhibit dynamic, time-dependent profiles of Gq and β-arrestin activity that in some cases exceed the activity of 5-HT (i.e., superagonism) at longer time points (e.g., 300 minutes, Supplementary Fig. 1A-H), which is not surprising given the dynamic temporal nature of GPCR signaling22. For all tested psychedelics, however, effects on Gq and β-arrestin activity were strikingly similar at equivalent time points and closely mirrored the pathway-balanced endogenous agonist 5-HT (Fig. 1L), indicating no strong preferences for one of the transducers. Psilocin, DMT and 2C-I showed slightly less β-arrestin2 efficacy compared to Gq activity, but the difference was not substantial when both transducers were compared at each respective time point (Supplementary Fig. 1B, C, E). Importantly, all tested psychedelics lacked a strong preference for Gq or β-arrestin2, demonstrating these compounds are not substantially biased for either transducer.
Rational design of a 5-HT2A-selective agonist template
To engineer a series of biased agonists for interrogating the 5-HT2AR-coupled signaling pathways associated with psychedelic potential, a scaffold exhibiting some degree of selectivity for 5-HT2AR over other 5-HT receptors is required. Most psychedelics are not selective for 5-HT2AR, exhibiting complex polypharmacology11. 5-HT2AR shares considerable homology with 5-HT2BR and 5-HT2CR, making it challenging to develop selective 5-HT2AR agonists. To date, few 5-HT2AR-selective agonists have been discovered, but the N-benzyl-phenethylamines 25CN-NBOH23 and DMBMPP24 are purported examples. Importantly, recently discovered “non-psychedelic” 5-HT2AR agonists show little selectivity for 5-HT2AR5,6, complicating attempts to interpret their psychopharmacology.
To develop selective 5-HT2AR biased ligands, we focused on the phenethylamine scaffold, which tends to have high selectivity for 5-HT2 subtypes. 25N or 2C-N (1) was selected as the core phenethylamine based on previous reports of potential 5-HT2AR biased agonism25,26. We confirmed 25N (1) is a high-affinity, potent 5-HT2 agonist with weak selectivity for 5-HT2AR and 5-HT2CR over 5-HT2BR (Fig. 2A, Supplementary Table 8). Because N-benzylation can increase affinity and potency of phenethylamines at 5-HT2AR27, we converted 25N (1) to 25N-NB (2) (see Supplementary Information), which reduced 5-HT2BR efficacy substantially (EMAX = 32% of 5-HT, Fig. 2A). Unfortunately, 25N-NB (2) retained potent 5-HT2CR activity (EC50 = 8.3 nM), resulting in a weakly (4-fold) 5-HT2AR-selective ligand.
Fig. 2: Rational design of a 5-HT2A-selective agonist template.
A N-Benzylation of 25N (1) to 25N-NB (2) leads to reduced 5-HT2B receptor efficacy, as measured by Gq dissociation by BRET. Data represent the mean and SEM from three independent experiments performed at 37 °C with 60-minute compound incubation. B Role of N-benzyl ring electrostatics in 5-HT2A receptor potency leading to development of 25N-NB-2-OH-3-Me (18) using QSAR correlation between 5-HT2A receptor pKi and Hammett σ constant values (Pearson’s R = −0.8887, R2 = 0.7897, 2-tailed p < 0.0001, N = 15). C The relationship between steric bulk and 5-HT2A/2C receptor selectivity is shown for the halogen series, leading to the identification of the 5-HT2A receptor-selective agonist 25N-NBI (10) (left). Also shown is a 5-HT2A/2C receptor selectivity heatmap comparing the 25N halogen series to 25CN-NBOH (right). D Comparison of 5-HT2A receptor (green) 5-HT2B receptor (red) and 5-HT2C receptor (purple) Gq dissociation activities for 25N-NBI (10) (left) and 25CN-NBOH (right). Data represent mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubation. E 5-HT2A receptor Gq dissociation and β-arrestin2 BRET concentration response curves for 25N-NBOH (3, top) and 25N-NB-2-OH-3-Me (18, bottom) showing addition of a 3-methyl group leads to reduced Gq-efficacy. Data represent mean and SEM from three independent experiments, which were performed at 37 °C with 60-minute compound incubation. F 25N-NBI (10) induced fit docking (IFD) with orthosteric site residue side chains displayed. The window shows a zoom-in view illustrating key ligand-residue interactions within the orthosteric site and illustrating the close proximity of the 2’- and 3’-positions to TM6 and TM7, which are known to influence ligand bias. G Summary of structure-activity relationships (SAR) for the 25N series encompassing…
Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential
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