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Structural pharmacology and therapeutic potential of 5-methoxytryptamines

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Abstract

Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders1,2,3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT2A (ref. 4). However, 5-HT1A 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-HT1A is a validated therapeutic target7,8, little is known about how psychedelics engage 5-HT1A 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-HT1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure–activity relationship analyses of 5-methoxytryptamines at both 5-HT1A and 5-HT2A enable the characterization of molecular determinants of 5-HT1A 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-HT1A agonists. We show that a 5-HT1A-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-HT1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.

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Fig. 1: Activity of psychedelics at 5-HT1A and 5-HT2A, and cryo-EM structures of 5-MeO-DMT and LSD bound to the 5-HT1A signalling complex.
Fig. 2: Differential pharmacological effects of amine and indole modifications to the 5-MeO-DMT scaffold at 5-HT1A and 5-HT2A.
Fig. 3: Elucidation of potency and selectivity determinants of tryptamines at 5-HT1A and 5-HT2A.
Fig. 4: Structural and functional comparison of 4-F,5-MeO-PyrT and clinical 5-HT1A medications at 5-HT1A.
Fig. 5: Effects of 5-MeO-DMT derivatives on mouse behaviour.

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Data availability

Density maps and structure coordinates have been deposited into the Electron Microscopy Data Bank (EMDB) and the PDB with the following accession identifiers: EMD-29560 and PDB 8FY8 for 5-MeO-DMT–5-HT1A–Gαi1–Gβ1–Gγ2; EMD-29597 and PDB 8FYT for LSD–5-HT1A–Gαi1–Gβ1-Gγ2; EMD-29571 and PDB 8FYE for 4-F,5-MeO-PyrT–5-HT1A–Gαi1–Gβ1–Gγ2; EMD-29585 and PDB 8FYL for vilazodone–5-HT1A–Gαi1–Gβ1–Gγ2; and EMD-29599 and PDB 8FYX for buspirone–5-HT1A–Gαi1–Gβ1–Gγ2.  Source data are provided with this paper. Additional data from this study are available upon request.

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Acknowledgements

This work was supported by NIH grant R35GM133504, a Sloan Research Fellowship in Neuroscience, an Edward Mallinckrodt, Jr Foundation Grant, a McKnight Foundation Scholars Award, an Irma T. Hirschl/Monique Weill-Caulier Trust Research Award (all to D.W.); an NIH F31 MH132317 (A.L.W), and T32 Training Grant GM062754 and DA053558 (A.L.W and G.Z.); the G. Harold & Leila Y. Mathers Charitable Foundation, the NIH grant R01DA050613, G.L. Freeman, and Columbia University for support of this work (all to D.S.); and the following NIH grants: R01MH127820 and R01MH104559 (S.J.R.). L.F.P is supported by the Leon Levy Foundation and the Brain and Behavior Research Foundation. Some of this work was performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539) and by grants from the Simons Foundation (SF349247) and NY State Assembly. We further acknowledge cryo-EM resources at the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310) with additional support from Agouron Institute (F00316) and NIH (OD019994). For additional data collection, we are grateful to staff at the Laboratory for BioMolecular Structure (LBMS), which is supported by the DOE Office of Biological and Environmental Research (KP160711). This work was supported in part through the computational and data resources and staff expertise provided by Scientific Computing and Data at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant ULTR004419 from the National Center for Advancing Translational Sciences. We thank B. Bechand for early examination of in vivo pharmacology of the described compounds assisted by V. C. Galicia; C. Hwu for assistance with synthesis and purification of several compounds (all at Columbia University); and F. Zandkarimi from the Columbia University Chemistry Department Mass Spectrometry Core Facility for conducting the high-resolution mass spectrometry experiments.

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  1. These authors contributed equally: Audrey L. Warren, David Lankri

Authors and Affiliations

  1. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Audrey L. Warren, Michael J. Capper & Daniel Wacker

  2. Department of Chemistry, Columbia University, New York, NY, USA

    David Lankri, Michael J. Cunningham, Inis C. Serrano, Andrew C. Kruegel, Priscilla Duggan, Vaclav Havel & Dalibor Sames

  3. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Lyonna F. Parise, Scott J. Russo & Daniel Wacker

  4. Zuckerman Institute of Mind, Brain, Behavior, Columbia University, New York, NY, USA

    Gregory Zilberg & Dalibor Sames

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Contributions

D.S., A.C.K, M. J. Cunningham and D.L. conceived and initiated the project. A.L.W. designed experiments, expressed and purified protein for grid freezing, collected data, refined structures with help from M. J. Capper, performed signalling and uptake assays, and co-wrote the manuscript. D.L. designed, synthesized, purified and characterized compounds with assistance from V.H., and co-wrote the manuscript. M. J. Cunningham designed, synthesized, purified and characterized compounds. D.L., V.H. and D.S. designed and supervised the pharmacokinetics study. L.F.P. performed the chronic SD stress assay and subsequent behavioural analyses supervised by S.J.R. I.C.S. designed and performed in vivo pharmacology assays, including the open-field and HTR assays, with assistance from P.D. G.Z. prepared grids for structure determination and assisted with data collection. D.S. and D.W. conceptualized the overall project and designed experiments, analysed the data, supervised the project and management, and wrote the manuscript.

Corresponding authors

Correspondence to Dalibor Sames or Daniel Wacker.

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Competing interests

The authors declare the following competing financial interests: D.S. and A.C.K. are co-founders of Gilgamesh Pharmaceuticals and Kures. M. J. Cunningham is a co-founder of Gilgamesh Pharmaceuticals. A.L.W., D.L., I.C.S., L.F.P., S.J.R., D.S. and D.W. are inventors on a patent application related to the featured compound class. D.W. has consulted for Otsuka Pharmaceutical, Longboard Pharmaceuticals and Ocean Bio on the design of psychedelic-based therapeutics. None of the companies listed herein contributed to the funding or experimental design. All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Cryo-EM structure determination of drug-bound 5-HT1A-Gαi1/Gβ1/Gγ2 complexes.

a, Representative structure determination of 5-MeO-DMT-bound 5-HT1A 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-HT1A-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-HT1A-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.

Source data

Extended Data Fig. 2 Comparison of different 5-HT1A structures and differences in binding of LSD to 5-HT1A and 5-HT2A.

a, Superposition of herein reported 5-MeO-DMT-bound 5-HT1A-Gi complex with the previously reported 5-HT-bound 5-HT1A-Gi structure (PDB ID: 7E2Y) shows similar conformations. Additional residues in 5-HT1A’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-HT1A-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-HT1A-Gi signalling complex and 5-HT2A (PDB ID: 6WGT). Left, 5-HT1A-bound LSD (grey) sits deeper in the binding pocket compared to 5-HT2A-bound LSD (orange). Zoom in of LSD in 5-HT1A-Gi structure (middle) and 5-HT2A structure (right) highlights differential stereochemistry and receptor-specific interactions of diethylamide moiety. Hydrogen bonds are indicated as grey dashed lines.

Source data

Extended Data Fig. 3 Global structure-activity landscape of tryptamine psychedelics at 5-HT1A and 5-HT2A receptors and their synthesis.

a, Overview of the cryo-EM structure of the 5HT1A 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-HT1A and 5-HT2A receptors (left semi-circle). 5-MeO-DMT (top of the circle), a major psychoactive compound found in toad secretions, shows comparable potency and efficacy at both 5-HT1A and 5-HT2A receptors. Systematic structural mapping via elaboration of the core 5-MeO-DMT structure identifies a class of 5-MeO-tryptamines with increasing 5-HT1A selectivity (right hemi-circle). 5-MeO-DMT can be viewed as a deconstruction of ibogaine, a oneirogen with a complex polycyclic tryptamine structure (bottom of the circle). Iboga compounds show no activity at 5-HT1A and 5-HT2A 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 work65. 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-HT1A.

a, Local resolution map of a 4-F,5-MeO-PyrT-bound 5-HT1A-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-HT1A 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-HT1A, 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.

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Extended Data Fig. 5 Comparison of 4-F,5-MeO-PyrT binding pose to that of different clinical 5-HT1A drugs.

a, b, Local resolution maps of vilazodone-bound (a) and buspirone-bound (b) 5-HT1A-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-HT1A’s orthosteric site.

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

a, 5-HT1A-Gi and 5-HT2A-Gq BRET of 5-HT, 5-MeO-DMT, 5-MeO-MET, and 4-F,5-MeO-PyrT with respective potencies and 5-HT1A > 5-HT2A 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-HT7A, 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.

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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 two-sided Fisher’s exact test and p value and number of mice have been denoted in the Figure. Error bars denote the s.e.m.

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Extended Data Table 1 Cryo-EM Data Collection, Model Refinement and Validation
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Extended Data Table 2 AUClast and Cmax of 4-F,5-MeO-PyrT at 1 mg/kg SC administration, and Plasma and Brain Tissue Binding of 4-F,5-MeO-PyrT
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Warren, A.L., Lankri, D., Cunningham, M.J. et al. Structural pharmacology and therapeutic potential of 5-methoxytryptamines. Nature (2024). https://doi.org/10.1038/s41586-024-07403-2

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