Structural insights into the unexpected agonism of tetracyclic antidepressants at serotonin receptors 5-HT1eR and 5-HT1FR

Abstract

Serotonin [5-hydroxytryptamine (5-HT)] acts via 13 different receptors in humans. Of these receptor subtypes, all but 5-HT_1eR have confirmed roles in native tissue and are validated drug targets. Despite 5-HT_1eR's therapeutic potential and plausible druggability, the mechanisms of its activation remain elusive. To illuminate 5-HT_1eR's pharmacology in relation to the highly homologous 5-HT_1FR, we screened a library of aminergic receptor ligands at both receptors and observe 5-HT_1eR/5-HT_1FR agonism by multicyclic drugs described as pan-antagonists at 5-HT receptors. Potent agonism by tetracyclic antidepressants mianserin, setiptiline, and mirtazapine suggests a mechanism for their clinically observed antimigraine properties. Using cryo-EM and mutagenesis studies, we uncover and characterize unique agonist-like binding poses of mianserin and setiptiline at 5-HT_1eR distinct from similar drug scaffolds in inactive-state 5-HTR structures. Together with computational studies, our data suggest that these binding poses alongside receptor-specific allosteric coupling in 5-HT_1eR and 5-HT_1FR contribute to the agonist activity of these antidepressants.

Fig. 1.. A curated screen of aminergic receptor ligands reveals 5-HT_1eR and 5-HT_1FR agonism by chemically diverse drugs and research chemicals.

(A) Activation of 5-HT_1eR and 5-HT_1FR as measured by cAMP accumulation and β-arrestin2 recruitment performed in human embryonic kidney (HEK) 293T cells. Experiments were performed in quadruplicate using 10 μM ligand concentration, and data are shown as log₂ fold change with compounds considered active when above 0.5 (dashed line). β-Arrestin2 recruitment data were normalized against DMSO, and cAMP accumulation data were normalized against effects in cells not transfected with receptor. For details, see table S1. (B) Venn diagram showing the number of compounds active in a single or multiple screens. (C) Two-dimensional (2D) chemical structures and respective concentration response curves in 5-HT_1eR– or 5-HT_1FR–mediated G_i1 protein dissociation in HEK293T cells. Experiments were performed in triplicate, and mean ± SEM from at least two (n = 2) independent experiments were averaged and normalized to 5-HT’s response. For details, see table S2.

Fig. 2.. Activities of multicyclic drugs at 5-HT_1eR and 5-HT_1FR.

2D chemical structures and respective concentration response curves in 5-HT_1eR– or 5-HT_1FR–mediated G_i1 protein dissociation in HEK293T cells are shown. Experiments were performed in triplicate, and mean ± SEM from at least two (n = 2) independent experiments were averaged and normalized to 5-HT’s response. For details, see table S2.

Fig. 3.. Cryo-EM structure determination of mianserin- and setiptiline-bound 5-HT_1eR–Gα_i1–Gβ₁–Gγ₂ complex.

(A) 2D chemical structures of structurally characterized 5-HT_1eR agonists (bottom) and overall structural model (left) and cryo-EM map (right) of mianserin-bound 5-HT_1eR–Gα_i1–Gβ₁–Gγ₂ complex. Mianserin, 5-HT_1eR, Gα_i1, Gβ₁, and Gγ₂ are colored in orange, green, violet, teal, and yellow. (B) Superposition of orthosteric ligand binding pockets of mianserin- and setiptiline-bound 5-HT_1eR. (C) Superposition of orthosteric ligand binding pockets of mianserin- and BRL-54443–bound 5-HT_1eR. Key side chains and drugs are shown as sticks, and a conserved salt bridge between drugs and D3.32 is shown as yellow dashed lines. Mianserin–5-HT_1eR complex is colored in orange and green, setiptiline–5-HT_1eR complex is colored in purple and pink, and BRL-54443–5-HT_1eR complex [Protein Data Bank (PDB): 7E33] is colored in dark blue and light blue.

Fig. 4.. Experimental and computational SAR studies of mianserin and setiptiline interactions with the 5-HT_1eR binding pocket.

(A) Views of the mianserin- and setiptiline-bound 5-HT_1eR orthosteric pocket, with key residues interacting with drugs shown as sticks. Conserved salt bridges between drugs and D3.32 are shown as yellow dashed lines. Mianserin–5-HT_1eR complex is colored in orange and green, setiptiline–5-HT_1eR complex is colored in purple and pink. (B) SIFt analyses indicate probabilities of ligand-receptor interactions formed during four 250-ns MD simulations of the mianserin/setiptiline–5-HT_1eR systems. The four interaction types formed by the ligands with the protein backbone and side chains are carbon-carbon atomic interactions (Apolar, pink), edge-to-face aromatic (Aro_E2F, light green), hydrogen bond with the protein as the hydrogen bond acceptor (Hbond_ProA, purple), and electrostatic interaction with the protein negatively charged (Elec_ProN, light green). Only interactions with an average probability greater than 10% are displayed. (C) Concentration response experiments determining the activity of 5-HT, mianserin, setiptiline, and BRL-54443 at wild type (WT) and mutant 5-HT_1eR as measured by G_i1 BRET in HEK293T cells. Experiments were performed in triplicate, and mean ± SEM from at least two (n = 2) independent experiments were averaged and normalized to 5-HT’s response. For details, see table S4.

Fig. 5.. Molecular basis of mianserin’s and setiptiline’s agonism at 5-HT_1eR.

(A) Superposition of mianserin-bound 5-HT_1eR and structures of inactive-state 5-HTRs bound to multicyclic antagonists reveals key differences in drug binding poses. Mianserin–5-HT_1eR complex is colored in orange and green, methiothepin–5-HT_1BR complex (PDB: 5V54) is colored in brown and yellow, and zotepine–5-HT_2AR complex (PDB: 6A94) is colored in pink and purple. Conserved ionic bonds between drugs and D3.32 are indicated as yellow dashed lines. Distances between mianserin and respective antagonists are measured between atoms closest to the 7TM core and shown as red arrows. 2D chemical structures of mianserin, methiothepin, and zotepine are shown on the right. (B) Shannon transfer entropy analysis between pairs of ligand-residue or residue-residue contacts within a minimum distance of less than 4.5 Å during MD simulations of mianserin–5-HT_1eR–G_i1 and setiptiline–5-HT_1eR–G_i1 complexes. Only residues that contribute markedly (>3%) to the transfer entropy between the ligand-binding pocket and the receptor G protein interface in each system are reported. (C) Sequence alignment of key residues of 5-HTR family involved in signal transduction.