Structural pharmacology and therapeutic potential of 5-methoxytryptamines

Abstract

Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders^1-3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT_2A (ref. ⁴). However, 5-HT_1A also plays a part in the behavioural effects of tryptamine hallucinogens⁵, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads⁶. Although 5-HT_1A is a validated therapeutic target^7,8, little is known about how psychedelics engage 5-HT_1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT_1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure-activity relationship analyses of 5-methoxytryptamines at both 5-HT_1A and 5-HT_2A enable the characterization of molecular determinants of 5-HT_1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT_1A agonists. We show that a 5-HT_1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT_1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.

Extended Data Fig. 1 |. Cryo-EM structure determination of drug-bound 5-HT_1A-Gαi1/Gβ1/Gβ2 complexes.

a, Representative structure determination of 5-MeO-DMT-bound 5-HT_1A signalling complex. Top Left, Analytical size exclusion chromatography and SDS-PAGE show monodisperse and pure protein of intact complex and its components. Right, representative Cryo-EM micrograph (white bar indicates scale) of 4680 total micrographs and data processing schematic exemplified by 5-MeO-DMT-bound 5-HT_1A-Gi1 structure: After particle picking, 2D classification and multiple rounds of 3D classification, the final particle stack was refined using non-uniform refinement. A final map was obtained and resolutions were estimated applying the 0.143 cutoff in GS-FSC. Initial models were built in COOT, and then further refined in PHENIX for the generation of final coordinates shown in this manuscript. b, Local resolution map of a 5-MeO-DMT-bound 5-HT_1A-Gi1 complex (left) and FSC curves (right) calculated based on the final reconstruction in cryoSPARC. c, 5-MeO-DMT (yellow) in the orthosteric binding pocket from the side (left) and rotated 45° towards the top of the receptor (right) with the map of ligand and surround residue densities shown at 5σ.

Extended Data Fig. 2 |. Comparison of different 5-HT_1A structures and differences in binding of LSD to 5-HT_1A and 5-HT_2A.

a, Superposition of herein reported 5-MeO-DMT-bound 5-HT_1A-Gi complex with the previously reported 5-HT-bound 5-HT_1A-Gi structure (PDB ID: 7E2Y) shows similar conformations. Additional residues in 5-HT_1A’s EL2 and G proteins not observed in previous structures are highlighted in red. b, Lipids (blue) and cholesterol hemisuccinate (dark blue) are bound to similar sites as observed before. c, Local resolution map of a LSD-bound 5-HT_1A-Gi1 complex (left) and FSC curves (right) calculated the final reconstruction in cryoSPARC. d, LSD (grey) in the orthosteric binding pocket from the side (top) and rotated 45° towards the top of the receptor (bottom) with the map of ligand and surround residue densities shown at 5σ. e, LSD shows distinct binding modes bound to 5-HT_1A-Gi signalling complex and 5-HT_2A (PDB ID: 6WGT). Left, 5-HT_1A-bound LSD (grey) sits deeper in the binding pocket compared to 5-HT_2A-bound LSD (orange). Zoom in of LSD in 5-HT_1A-Gi structure (middle) and 5-HT_2A structure (right) highlights differential stereochemistry and receptor-specific interactions of diethylamide moiety. Hydrogen bonds are indicated as grey dashed lines.

Extended Data Fig. 3 |. Global structure-activity landscape of tryptamine psychedelics at 5-HT_1A and 5-HT_2A receptors and their synthesis.

a, Overview of the cryo-EM structure of the 5HT_1A receptor-Gi signalling complex bound to 5-MeO-DMT (center). Classic psychedelics such as the prototypical compounds DMT and LSD are agonists of both 5-HT_1A and 5-HT_2A receptors (left semicircle). 5-MeO-DMT (top ofthe circle), a major psychoactive compound found in toad secretions, shows comparable potency and efficacy at both 5-HT_1A and 5-HT_2A receptors. Systematic structural mapping via elaboration of the core 5-MeO-DMT structure identifies a class of 5-MeO-tryptamines with increasing 5-HT_1A selectivity (right hemi-circle). 5-MeO-DMT can be viewed as a deconstruction of ibogaine, a oneirogen with a complex polycyclic tryptamine structure (bottom ofthe circle). Iboga compounds show no activity at 5-HT_1A and 5-HT_2A receptors, but this activity re-emerges by deconstruction of the isoquinuclidine core to simple mono-cyclic tryptamines such as 5-MeO-PipT (5-methoxypiperidinyl-tryptamine) and 4-F,5-MeO-PyrT (4-fluoro, 5-methoxypyrrolidinyl-tryptamine, right hemi-circle). Images of peyote, mushrooms, ayahuasca, and toad are from iStock and ShutterStock, and Tabernanthe iboga schematic is adapted from previous work. b, General synthesis methodology of tryptamines. a. oxalyl chloride, b. MeOH, LAH, c. PPh3, CBr4, d. Amine, TEA, e. Amine, f. LAH.

Extended Data Fig. 4 |. Structural comparison of 5-MeO-DMT 5-HT1A-selective analog 4-F, 5-MeO-PyrT bound to 5-HT_1A.

a, Local resolution map of a 4-F,5-MeO-PyrT-bound 5-HT_1A-Gi1 complex (left) and FSC curves (right) calculated from the final reconstruction in cryoSPARC. b, 4-F,5-MeO-PyrT (dark blue) in the orthosteric binding pocket from the side (left) and rotated 45° towards the top of the receptor (right) with the map of ligand and surrounding residue densities shown at 5σ. c, structural side-by-side comparison of 5-HT_1A orthosteric site bound to 5-MeO-DMT (yellow) and 4-F,5-MeO-PyrT (dark blue). d, cAMP accumulation assays using wildtype and mutant 5-HT_1A, and different drugs. Concentration-response experiments reveal different sensitivities of distinct drugs to F361L mutation. All signalling studies were performed in triplicates and are averaged from two to three independent experiments. Data have been normalized against 5-HT and errors bars denote s.e.m.

Extended Data Fig. 5 |. Comparison of 4-F,5-MeO-PyrT binding pose to that of different clinical 5-HT_1A drugs.

a, b, Local resolution maps of vilazodone-bound (a) and buspirone-bound (b) 5-HT_1A-Gi1 complexes and corresponding FSC curves calculated from the final reconstructions in cryoSPARC. c, d, Vilazodone (c, green) and buspirone (d, teal) in the orthosteric binding pocket from the side (left) and rotated 45° towards the top of the receptor (right) with the density map of ligand and surrounding residues shown at 5σ. e-h, Extracellular view of 4-F,5-MeO-PyrT (e, dark blue), Vilazodone (f, green), Aripiprazole (g, magenta, PDB ID: 7E2Z), and Buspirone (h, teal) binding poses in 5-HT_1A’s orthosteric site.

Extended Data Fig. 6 |. Selectivity of different 5-MeO-DMT analogs and off-target activity of 4-F,5-MeO-PyrT.

a, 5-HT_1A-Gi and 5-HT_2A-Gq BRET of 5-HT, 5-MeO-DMT, 5-MeO-MET, and 4-F,5-MeO-PyrT with respective potencies and 5-HT_1A >5-HT_2A selectivities. b, Off-target inhibition of transporters SERT, PMAT, OCT1, and OCT2 by 4-F,5-MeO-PyrT and known inhibitors. SERT uptake was performed in triplicates and data was averaged from two independent experiments showing data as mean+s.e.m. PMAT, OCT1, and OCT2 uptake was performed once in quadruplicate. c, Arrestin-recruitment of 5-HT and 4-F,5-MeO-PyrT at all human 5-HT receptor subtypes except for 5-HT_7A, whose activation was measured via cAMP stimulation. All functional studies were performed in triplicates and are averaged from two to three independent experiments. Data have been normalized against 5-HT, Citalopram, and Decynium-22, and errors bars denote the s.e.m.

Extended Data Fig. 7 |. Effects of 5-MeO-DMT derivatives on rodent behavior.

a, Evaluation of 4-F,5-MeO-PyrT in open field for two hours (n = 3-4/group). b, Exemplary traces of the ambulatory distance traveled in open field following 4-F,5-MeO-PyrT (1 mg/kg, s.c.) administration and with and without WAY-100635 pre-treatment (1 mg/kg, s.c., 15 min prior). Panel was created with BioRender.com. c, Effect of WAY-100635 (1 mg/kg, s.c., 15 min prior) on 4-F,5-MeO-PyrT’s and 5-MeO-MET’s effect on total locomotion (n = 7-8/group, 30 min). d, Determination of optimal inhibitory WAY-100635 dose via administration of 1 mg/kg and 2 mg/kg WAY-100635 prior to studying 4-F,5-MeO-PyrT’s effects on total locomotion (n = 7 - 8/group) and HTR (n = 6/group). Analysis was done using one-way ANOVA with multiple comparisons with Tukey’s post hoc test, and exact p values have been denoted in the Figure. e-g, Effects of saline or 4-F,5-MeO-PyrT administration on control (Control) or chronically defeated mice (Stress). Determination of e, distance moved as a measure of locomotor activity, f, social interaction as a measure of anxiety- and depression-related phenotype, g, corner time as a measure of anxiety-like behavior. Analysis was done in a sub-cohort of the animals reported in Fig. 5d. Number of mice for each group is indicated below the data for each respective cohort. Differences were determined by two-way ANOVA with multiple comparisons using Fisher’s LSD post hoc test, and exact p values have been denoted in the Figure. h, Vehicle- and drug-treated stressed mice shown in Fig. 5d were divided into susceptible (SI ratio<1) and resilient (SI ratio>1) populations. Significance in the population shift was determined by a twosided Fisher’s exact test and p value and number of mice have been denoted in the Figure. Error bars denote the s.e.m.

Fig. 1 |. Activity of psychedelics at 5-HT_1A and 5-HT_2A, and cryo-EM structures of 5-MeO-DMT and LSD bound to the 5-HT_1A signalling complex.

a, 5-HT_1A-mediated G_i1 activation (blue) and 5-HT_2A-mediated G_q activation (red) by psychedelics determined by BRET. Concentration–response experiments were performed in triplicate and are averaged from two (mescaline) or more (all other compounds) independent experiments. Data are normalized against 5-HT and errors bars denote the s.e.m. b, Top, cryo-EM structure of 5-HT_1A–Gα_i1–Gβ₁-Gγ₂ signalling complexes. 5-HT_1A, Gα_i1, Gβ₁ and Gγ₂ are shown in pink, dark blue, teal and yellow, respectively. Middle, zoom-in images show the 5-HT_1A orthosteric site bound to 5-MeO-DMT (yellow) and LSD (grey), with ionic interactions and hydrogen bonds indicated by dashed lines. Bottom, superposition of 5-HT_1A orthosteric binding sites comparing binding poses of 5-MeO-DMT and LSD, as well as 5-MeO-DMT and 5-HT (Protein Data Bank (PDB) identifier: 7E2Y).

Fig. 2 |. Differential pharmacological effects of amine and indole modifications to the 5-MeO-DMT scaffold at 5-HT_1A and 5-HT_2A.

a,b, Schematic of modifications (left) and 5-HT_1A–Gi and 5-HT_2A–G_q BRET concentration–response activity (right) mediated by ‘designer’ tryptamines (a) and cyclized tryptamines (b). c,d, Schematic of modifications (left) and effects of indole modifications at the fifth position (c) and 4-fluorination (d) on 5-HT_1A–Gi and 5-HT_2A–G_q BRET activity (right). All signalling experiments were performed in triplicate and are averaged from two (5-MeO-MiPT, 5-MeO-3-PyrrolineT, 5-MeO-PipT) or three (all other compounds) independent experiments. Data are normalized against 5-HT and errors bars denote the s.e.m.

Fig. 3 |. Elucidation of potency and selectivity determinants of tryptamines at 5-HT_1A and 5-HT_2A.

a, Left and right, 5-HT_1A–G_i and 5-HT_2A–G_q signalling mediated by tryptamine compounds at mutant and wild-type (WT) receptors according to BRET assays. Middle, residue differences in OBPs of 5-HT_1A (pink) and 5-HT_2A (green; PDB identifier 6WGT). b, Heatmap showing the effect of tryptamine modifications on the signalling potency (pEC₅₀) at mutant and wild-type 5-HT_1A and 5-HT_2A receptors. Grey boxes indicate compound not tested. All signalling experiments were performed in triplicate and are averaged from two (5-HT_1A(N386V), 5-HT_2A(V366N), 5-HT_1A(A365N): 4-F,5-MeO-PyrT; 5-HT_2A(N343A): 4-F,5-MeO-PyrT) or three (all other compounds) independent experiments. Data are normalized against 5-HT and errors bars denote the s.e.m.

Fig. 4 |. Structural and functional comparison of 4-F,5-MeO-PyrT and clinical 5-HT_1A medications at 5-HT_1A.

a, Top, two-dimensional structures of 4-F,5-MeO-PyrT, vilazodone, aripiprazole and buspirone. Bottom, structural comparison of drug-binding poses of 4-F,5-MeO-PyrT (blue), vilazodone (green) and buspirone (teal) determined in this study, as well as a previous structure of aripiprazole (magenta; PDB identifier 7E2Z). Buspirone assumes a kinked conformation binding to a previously undescribed EBP (EBP2, blue shade), whereas vilazodone and aripiprazole stretch towards the extracellular space forming interactions in a distinct EBP (EBP1, yellow shade). All compounds assume similar overall poses in the OBP (grey shade) of 5-HT_1A. b, 5-HT_1A–G_i BRET values of 4-F,5-MeO-PyrT, vilazodone, aripiprazole and buspirone. All signalling experiments were performed in triplicate and are averaged from two (aripiprazole) or three (all other compounds) independent experiments. Data are normalized against 5-HT and errors bars denote the s.e.m.

Fig. 5 |. Effects of 5-MeO-DMT derivatives on mouse behaviour.

a, Pharmacokinetics profile of 4-F,5-MeO-PyrT following s.c. administration of 1 mg kg⁻¹. Data were obtained from three mice per time point (n = 3). b, HTR as a measure of 5-HT_2A-mediated hallucinogenic activity in mice in the presence and absence of the 5-HT_1A-selective antagonist WAY-100635 (1 mg kg⁻¹). c, Schematic of the chronic SD stress paradigm. d,e, Effects of saline, 4-F,5-MeO-PyrT and WAY-100635 administration on control mice and chronically defeated (stressed) mice. Determination of SI (d) and preference for 1% sucrose (SUC) solution over water in a two-bottle choice test (e) as a measure of a depressive-like phenotype. Compounds were dosed at 1 mg kg⁻¹ unless otherwise indicated. Data are averaged from three (d) and two (e) independent experiments, and the number of mice for each group is indicated below the data for each respective group. Differences were determined by two-way analysis of variance with multiple comparisons using Fisher’s least significance difference post hoc test, and exact P values are denoted in the figure. Error bars denote the s.e.m. Schematic in c was created using BioRender (https://www.biorender.com).