Structure-Activity Assessment and In-Depth Analysis of Biased Agonism in a Set of Phenylalkylamine 5-HT2A Receptor Agonists

Abstract

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 EC₅₀ and E_max 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_q- 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_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).

Keywords: 5-HT2A; biased agonism; in vitro pharmacology; miniGαq; psychedelics; β-arrestin.

Figure 1

Schematic depiction of the structures of the phenylalkylamine analogues assessed in the present study, with phenethylamines (2C-X) highlighted in gray, phenylisopropylamines (DOX) highlighted in yellow, and N-benzylphenethylamines (25X-NB) highlighted in blue. Orange arrows denote the structural comparisons of compounds that only differ in terms of their 4-position substituent, green arrows indicate the comparisons of 2C-X analogues with the corresponding DOX analogues, and blue arrows indicate the comparison between 2C-X analogues and their N-benzyl-derived counterparts. The R₂ and R₃ substituents are consistent with Scheme 1.

Figure 2

(a–c) Predicted binding poses and ligand–receptor interactions of selected 4-substituted phenethylamines at the 5-HT_2A. The ligands are displayed as sticks, while the receptor is shown as gray lines and cartoon. Ligand–receptor interactions are displayed as dashed lines and colored in green (aromatic, π–π stacking), yellow (hydrogen bond), and pink (salt-bridge). The receptor hydrophobic sub-pocket between TM4 and TM5 is shown as a surface and colored according to the Eisenberg hydrophobicity scale, from highly hydrophilic (blue) to highly hydrophobic (yellow). (d) Linear correlation curve between the miniGα_q (blue) and βarr2 (orange) recruitment assay E_max values and calculated c log D values at pH 7.40.

Figure 3

Concentration–response curves for each of the compounds in the βarr2 (orange) and miniGα_q (blue) recruitment assays at the 5-HT_2A. Each point represents the mean of three independent experiments, each performed in duplicate ± SEM (standard error of the mean). Curves represent three parametric, nonlinear fits of normalized and pooled data for each concentration tested, normalized with LSD as a reference agonist.

Figure 4

Visual representation of the bias factors (β) ± SEM (standard error of the mean), where ** stands for p < 0.01 in the nonparametric Kruskal–Wallis analysis of significance with post hoc Dunn’s test. LSD is used as the reference agonist.

Figure 5

Qualitative bias plots, where each panel shows the centered second-order polynomial fit of the activation values at equimolar concentrations of the substance in the respective assays in red, and that of the reference agonist (in this case LSD) in black.

Figure 6

Visual representation of the differences in ranking order of the substances’ efficacies in the βarr2 and miniGα_q assay at the 5-HT_2A. The x-axis shows the relative ranking of the substance in the βarr2 assay, and the y-axis shows that in the miniGα_q assay. The substances that are on the right part of the “identity line” have a relative preference toward miniGα_q recruitment, and the ones above the line toward βarr2 recruitment, with substances being further away from the unity line having a larger difference in ranking order.

Scheme 1. Synthesis of the Phenylalkylamines and Their Corresponding N-Benzyl Analogues

Reaction conditions: (a) (i) aldehyde, EtOH, rt; (ii) NaBH₄, EtOH, rt; (b) (i) phthalic anhydride, toluene, reflux; (ii) TiCl₄, dichloromethyl methyl ether/4-chlorobutanoyl chloride, CH₂Cl₂, −10 to 0 °C; (iii) NaBH₄, EtOH, rt; (iv) NaH, EtI, DMF, 0 °C to rt; (v) hydrazine (aq.), THF, rt; (c) HNO₃ (70%), AcOH, 0 °C–rt. (d) (i) Br₂, AcOH, rt; (ii) phthalic anhydride, toluene, reflux; (iii) Cu(I)CN, DMF, reflux; (iv) hydrazine (aq.), THF, rt.