Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential

Abstract

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT_2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT_2A-Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT_2A-selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT_2A-Gq but not 5-HT_2A-β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT_2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT_2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT_2A agonists. We also demonstrate that β-arrestin-biased 5-HT_2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT_2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.

Fig. 1. Psychedelics exhibit similar Gq and β-arrestin2 activity at 5-HT_2AR.

Schematic of 5-HT_2A receptor G protein dissociation (A) and β-arrestin recruitment (B) to determine transducer preferences. (C) Determination of 5-HT_2A 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-HT_2A 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 (E_MAX/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.

Fig. 2. Rational design of a 5-HT_2A-selective agonist template.

A N-Benzylation of 25N (1) to 25N-NB (2) leads to reduced 5-HT_2B 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-HT_2A receptor potency leading to development of 25N-NB-2-OH-3-Me (18) using QSAR correlation between 5-HT_2A receptor pK_i 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-HT_2A/2C receptor selectivity is shown for the halogen series, leading to the identification of the 5-HT_2A receptor-selective agonist 25N-NBI (10) (left). Also shown is a 5-HT_2A/2C receptor selectivity heatmap comparing the 25N halogen series to 25CN-NBOH (right). D Comparison of 5-HT_2A receptor (green) 5-HT_2B receptor (red) and 5-HT_2C 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-HT_2A 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 key effects on electrostatics, 5-HT_2A receptor selectivity, and reduced Gq E_MAX. Source data are provided as a Source Data file.

Fig. 3. Structure-based design of β-arrestin-biased 5-HT_2A agonists.

A Effect of the larger N-substituted 25N analogs 25N-N1-Nap (16) and 25N-NBPh (17) on 5-HT_2A receptor Gq dissociation (red) and β-arrestin2 (blue) recruitment. Data represent the mean and SEM from three independent experiments performed at 37 °C with 60-minute compound incubation. B Outward pivot of TM6 for 25CN-NBOH and 25N-N1-Nap (16) MD simulations relative to the inactive 5-HT_2A receptor structure with final frame shown as representative. Note the intermediate TM6 tilt angle with 25N-N1-Nap (16) relative to that of 25CN-NBOH. C Change in W366^6.48 toggle switch χ² angle. χ² angle given is the absolute difference in peak angle relative to the inactive state. Final frame shown as representative. D Distribution and time dependence W366^6.48 χ² angle of 25CN-NBOH (yellow) and 25N-N1-Nap (16) (orange) simulations. E Effect of 5-HT_2A receptor W336^6.48Y mutation on 25N-NBOMe (4), 25N-NBPh (17), and 25N-N1-Nap (16) Gq dissociation (red) versus β-arrestin2 (blue) recruitment activities. Data represent the mean and SEM from three independent experiments performed at 37 °C with 60-minute compound incubation. Source data are provided as a Source Data file.

Fig. 4. β-Arrestin-biased 5-HT_2A agonists lack psychedelic potential.

A 25N-NBOMe (4) induces the head-twitch response (HTR) (W_5,19.46 = 84.56, p < 0.0001). B 25N-N1-Nap (16) does not induce the HTR (F_5,26 = 1.39, p = 0.2618). C 25N-NBPh (17) does not induce the HTR (F_4,18 = 0.89, p = 0.4903). D 25N-NB-2-OH-3-Me (18) does not induce the HTR (F_2,25 = 0.55, p = 0.7377). E Illustration showing the procedures used to confirm that compounds tested in panels F–H are brain penetrant and capable of engaging 5-HT_2A receptors in the CNS of mice. Mice were pretreated with vehicle or drug, ( ± )-DOI (1 mg/kg IP) was injected 10 minutes later, and then HTR activity was assessed for 20 minutes. F Pretreatment with 25N-N1-Nap (16) blocks the HTR induced by (±)-DOI (F_3,16 = 68.43, p < 0.0001). G Pretreatment with 25N-NBPh (17) blocks the HTR induced by (±)-DOI (W_3,14.64 = 144.6, p < 0.0001). H Pretreatment with 25N-NB-2-OH-3-Me (18) blocks the HTR induced by (±)-DOI (F_3,16 = 18.39, p < 0.0001). P values are provided if there were significant differences between groups (Tukey’s test or Dunnett’s T3 test). HTR counts from individual male C57BL/6 J mice as well as group means are shown. Drug doses are presented as mg/kg. I 5-HT_2A receptor Gq-mediated calcium flux activity comparing 5-HT (open circles) antagonist activity for 25N-N1-Nap (16) (red), 25N-NB-2-OH-3-Me (18) to M100,907 (black circles). Data represent the mean and SEM from three independent experiments. Source data are provided as a Source Data file.

Fig. 5. Psychedelic potential is correlated with 5-HT_2A-Gq signaling.

A Scatter plot showing the relationship between 5-HT_2A receptor Gq-efficacy (E_MAX values) and head-twitch response (HTR) magnitude (maximum counts per minute induced by the compound) for 14 members of the 25N series. Spearman’s rank correlation coefficient R_s is shown. The regression line generated by fitting the data using non-linear regression is included as a visual aid. B Scatter plot showing the relationship between 5-HT_2A receptor β-arrestin2 recruitment efficacy (E_MAX values) and HTR magnitude for 14 members of the 25N series. C Scatter plot showing the relationship between 5-HT_2A receptor Gq-efficacy (E_MAX values) and HTR magnitude for 24 phenethylamine psychedelics. To illustrate the non-linear nature of the relationship, the regression line generated by fitting the data using non-linear regression is shown. D Scatter plot showing the relationship between 5-HT_2A receptor β-arrestin2 recruitment efficacy (E_MAX values) and HTR magnitude for 24 phenethylamine psychedelics. Source data are provided as a Source Data file.

Fig. 6. 5-HT_2A-Gq signaling predicts psychedelic potential.

A–G Activity in the HTR assay can be predicted based on 5-HT_2A receptor Gq-efficacy. As predicted, 25O-NBOMe (32) (F_5,26 = 37.01, p < 0.0001), 2C2-NBOMe (31) (W_6,12.67 = 96.28, p < 0.0001), and 25O-NBcP (33) (F_5,25 = 20.57, p < 0.0001) induced head twitches, whereas 25O-NBPh-10’-OH (35) (F_4,27 = 2.58, p = 0.0601), 25O-NB-3-I (34) (F_5,25 = 0.26, p = 0.9288), and 25D-N1-Nap (26) (F_3,20 = 0.37, p = 0.7748) did not induce head twitches. (H) Pretreatment with the Gαq/11 inhibitor YM-254890 blocks the HTR induced by R-(-)-DOI (F_3,12 = 8.69, p = 0.0025). Mice were treated ICV with YM-254890 or vehicle and then all of the animals received R-(-)-DOI (1 mg/kg IP) 15 minutes later. (I) Pretreatment with the phospholipase C (PLC) inhibitor edelfosine blocks the HTR induced by R-(-)-DOI (F_2,16 = 11.34, p = 0.0009) and 25N-NBOMe (4) (F_2,16 = 6.40, p = 0.0091). Mice received three consecutive injections of vehicle or the indicated dose of edelfosine at 20-minute intervals and then a HTR-inducing drug was administered 10 minutes after the third injection. HTR counts from individual male C57BL/6 J mice as well as group means are shown. P values are provided if there were significant differences vs. vehicle control (Tukey’s test or Dunnett’s T3 test). The dose of YM-254890 is presented in µg; other drug doses are presented as mg/kg. Source data are provided as a Source Data file.

Fig. 7. Non-psychedelics do not achieve 5-HT_2A Gq-signaling efficacy threshold.

A Effect of (+)-lysergic acid diethylamide (LSD), psilocin, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), N,N-diethyltryptamine (DET), lisuride, ( + )−2-bromolysergic acid diethylamide (2-Br-LSD), 6-methoxy-N,N-dimethyltryptamine (6-MeO-DMT), and 6-fluoro-N,N-diethyltryptamine (6-F-DET) on 5-HT_2A receptor Gq dissociation (red) and β-arrestin2 (blue) recruitment. The BRET data for LSD, psilocin, and 5-MeO-DMT are from Fig. 1; the data for 2-Br-LSD were published previously. Data represent the mean and SEM from three independent experiments. B Effect of LSD, psilocin, 5-MeO-DMT (F_4,26 = 3.09, p = 0.0331), DET, lisuride, 2-Br-LSD, 6-MeO-DMT (F_5,26 = 4.19, p = 0.0063), and 6-F-DET (F_4,23 = 13.49, p < 0.0001) on the head-twitch response (HTR) in mice. The HTR data for LSD, DET, psilocin, lisuride, and 2-Br-LSD were re-analyzed from published experiments^,,,. HTR counts from individual male C57BL/6 J mice as well as group means are shown. P-values are provided if there were significant differences between groups (Tukey’s test). C Scatter plot and linear regression showing the correlation between 5-HT_2A receptor Gq-efficacy (E_MAX values) and HTR magnitude (maximum counts per minute induced by each drug). Pearson’s correlation coefficient R is shown. The p-value is 2-tailed. Drug doses are presented as mg/kg. Source data are provided as a Source Data file.

Fig. 8. In vitro and in vivo effects of β-arrestin-biased 5-HT_2A agonists.

A Scheme of NanoBit Internalization Assay for 5-HT_2AR measuring loss of surface expression with the membrane impermeable LgBit. B 5-HT_2AR internalization concentration response calculated as percent basal surface expression and normalized to 0% of max 5-HT response. Data represent mean and SEM from 3 independent experiments with 5-HT (black), DOI (blue), pimavanserin (grey), 25N-NBOMe (4) (green), 25N-N1-Nap (16) (red) and 25N-NBPh (17) (purple). C Cartoon showing the procedures used to induce tolerance in the head-twitch response (HTR) assay. Mice were injected with vehicle or drug once daily for 5 consecutive days and then challenged with (±)-DOI (1 mg/kg IP) 24 hours after the last injection. D Repeated administration of (±)-DOI and 25N-N1-Nap (16) (F_2,16 = 16.68, p = 0.0001), but not pimavanserin (t₁₀ = 1.067, 2-tailed p = 0.3111), induces a tolerance to the HTR induced by DOI. Mice was treated with vehicle, 25N-N1-Nap (16) (20 mg/kg/day SC), or (±)-DOI (10 mg/kg/day SC). Additional mice were treated with vehicle or pimavanserin (1 mg/kg/day SC). HTR counts are expressed as a percentage of the response in the respective control group; data from individual male C57BL/6 J mice as well as group means are shown. P-values are provided if there were significant differences between groups (Tukey’s test). E Pimavanserin blocks the response to (±)-DOI injected 20-minutes later (F_3,16 = 8.19, p = 0.0016). HTR counts from individual mice as well as group means are shown. P values are provided if there were significant differences between groups (Tukey’s test). F 25N-N1-Nap (16) attenuates phencyclidine (PCP)-induced hyperactivity (pretreatment × PCP: F_1,20 = 7.09, p = 0.015; pretreatment × PCP × time: F_2,40 = 11.03, p = 0.0002). Locomotor activity was measured as distance traveled (cm), presented as group means ± SEM. N = 6 male C57BL/6 J mice/group. *p < 0.05, significant difference vs. vehicle; ^#p < 0.05, significant difference vs. PCP alone (Tukey’s test). G M100907 attenuates PCP-induced hyperactivity (pretreatment × PCP: F_1,20 = 7.41, p = 0.0131). N = 6 male C57BL/6 J mice/group. *p < 0.05, significant difference vs. vehicle; ^#p < 0.05, significant difference vs. PCP alone (Tukey’s test). Drug doses are presented as mg/kg. Source data are provided as a Source Data file.