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Structural determinants for activity of the antidepressant vortioxetine at human and rodent 5-HT3 receptors

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug’s molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.

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Fig. 1: VTX acts as a partial agonist at human receptors and as an antagonist at rodent receptors.
Fig. 2: Structure and conformation of the mouse 5-HT3A receptor in complex with VTX.
Fig. 3: Structures and conformations of the human 5-HT3A receptor.
Fig. 4: Structural and functional data support the hypothesis that VTX acts through desensitization in h5-HT3AR.
Fig. 5: Comparison of VTX binding at m5-HT3AR and h5-HT3AR.

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Data availability

The coordinates of the structures and the cryo-EM maps have been deposited with the Worldwide Protein Data Bank (wwPDB) and Electron Microscopy Data Bank (EMDB): m5-HT3AR in detergent in complex with VTX (EMDB-15689, PDB 8AW2), h5-HT3AR in detergent apo/resting (EMDB-15699, PDB 8AXD), h5-HT3AR in detergent active-distorted (EMDB-16103, PDB 8BL8), h5-HT3AR in detergent (EMDB-16104, PDB 8BLA), h5-HT3AR in nanodisc in complex with VTX (EMDB-16105, PDB 8BLB). All relevant data have been deposited in publicly available repositories. Simulation files have been deposited in Zenodo at https://doi.org/10.5281/zenodo.10663688. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Danish Council of Independent Research for Medical Sciences (grant number DFF-404-00309, A.S.K.), the Lundbeck Foundation (grant number 2017-1655 and 2012-12453, A.S.K.), the Carlsberg Foundation (A.S.K.), the ERC Starting grant 637733 Pentabrain (HN), the Fondation pour la Recherche Médicale (grant number SPF201809007073, U.L.S.), the State-Region Plan “Technological Innovations, Modeling and Personalized Medical Support” (IT2MP, to F.D.) and the European Regional Development Funds (ERDF, to F.D.). Computations were made possible through allocations at the Centre for Scientific Computing, Aarhus (SCS-Aa). We acknowledge access to the ESRF CM01 Krios microscope. The work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The electron microscopy facility is supported by the Rhône-Alpes Region, the FRM, the FEDER, and the GIS-IBISA. We thank L. Zarkadas for his help with cryoEM.

Author information

Author notes

  1. Uriel López-Sánchez

    Present address: Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

  2. These authors contributed equally: Uriel López-Sánchez, Lachlan Jake Munro.

Authors and Affiliations

  1. University Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France

    Uriel López-Sánchez, Delphine Baud, Céline Juillan-Binard, Guy Schoehn, Jacques Neyton & Hugues Nury

  2. Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark

    Lachlan Jake Munro, Anders Juel Pedersen, Christian Colding Brun, Signe Meisner Lyngby, Miriam Grønborg Pedersen & Anders S. Kristensen

  3. Department of Chemistry, Aarhus University, Aarhus, Denmark

    Lucy Kate Ladefoged & Birgit Schiøtt

  4. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Sarah C. R. Lummis

  5. Lundbeck Research, H. Lundbeck A/S, Valby, Denmark

    Benny Bang-Andersen

  6. Université de Lorraine, CNRS, LPCT, Nancy, France

    Christophe Chipot & Francois Dehez

  7. Laboratoire International Associé CNRS and University of Illinois at Urbana-Champaign, Vandoeuvre-les-Nancy, France

    Christophe Chipot & Francois Dehez

  8. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Christophe Chipot

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Contributions

All authors participated in research design. U.L.-S., L.J.M., D.B., L.K.L., A.J.P., Pedersen, S.C.R.L., C.C.B., S.M.L., G.S., J.N., F.D., C.C., H.N. and A.S.K. conducted experiments. B.B.-A. contributed new reagents and analytical tools. U.L.S., L.J.M., L.K.L., M.G.P., S.C.R.L., C.C.B., S.M.L., B.S., J.N., F.D., H.N. and A.S.K. performed data analysis. U.L.S., L.J.M., L.K.L., F.D., J.N., H.N. and A.S.K. wrote or contributed to the writing of the manuscript. All authors reviewed the final manuscript.

Corresponding authors

Correspondence to Hugues Nury or Anders S. Kristensen.

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B.A. is an employee and shareholder of Lundbeck A/S. The remaining authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Jeff Abramson and Rebecca Howard for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Nomenclature and sequence alignment.

a, Ribbon illustration of a full 5-HT3R subunit and the extracellular domain of a complementary subunit. Binding elements are indicated in colors: Loops A-C on the principal subunit, three strands and a loop D-G on the complementary subunit. b, Multiple sequence alignment of the mouse, human, rat, and guinea pig 5-HT3A receptors. Residues of interest in Loops F and C are shown in bold. c, Amino-acid percent identity matrix of mouse, human, rat and guinea 5-HT3A receptors.

Extended Data Fig. 2 Quality of the density maps.

a, m5-HT3AR, b, apo resting h5-HT3AR, c, apo active-distorted h5-HT3AR, d, h5-HT3AR in complex with VTX (detergent) and e, h5-HT3AR in complex with VTX (nanodisc) reconstructions in surface representation overlaid with the structures. From left to right: densities of the β-sheets in the ECD, densities of the Cys-loop and/or the M2–M3 loop, densities of helices M1 and M2, densities of M3 and M4, densities of M2 at the level of L9′ (L260).

Extended Data Fig. 3 Image analysis workflow, m5-HT3AR in detergent dataset.

a, Schematic of the image analysis workflow b, Example of one micrograph from the 3000 in the dataset. c, Selected 2D class averages of the final particles set. d, Side view of the final reconstruction. The sharpened 3D density map is colored according to the local resolution (FSC threshold of 0.143). e, Heat map of the angular distribution of particle projections for the reconstruction. f, Gold-standard FSC curves. The dotted line represents the 0.143 FSC threshold.

Extended Data Fig. 4 Comparative features of the m5-HT3AR and h5-HT3AR during molecular dynamics simulations.

a-b, Probability density functions (PDFs) for the surface area of the inter-subunit ECD-ECD interfaces (a) and loop C to loop D distance (b) the for human (blue) and mouse (green) during the MD simulations receptors. Loop C-D distance is calculated between center-of-mass of Ca atoms in residues 222-228 in loop C and Cα of R87 of loop D. Fitting of a Gaussian mixture model (dashed lines) were used to determine Gaussian components using the Bayesian information criterion. c, RMSF for ring A-C. Bar graphs represent the mean ± SEM (n = 25 samples). d, Time course of the RMSD of Cα atoms of the TMDs (pale colors) and the ECDs (dark colors) showing larger deviations for the human receptor TMD. e, Probability of hydrogen bond or cation/π interaction between the amine group in VTX and nearby residues N123, S177, and W178 (human residue numbering) in the simulations of human (Ph) and mouse (Pm) 5-HT3AR. f, Probability of the ring A phenyl group assuming upwards- or downwards-facing orientations. g, Average pore profile during the initial 50 ns MD simulations with backbone restraints in h5-HT3AR (dark blue) and m5-HT3AR (dark green) compared to the profiles observed during the final 50 ns without restraints (light blue and green, respectively) in the five individual simulations. Error bars represent the standard deviation. h, Time progression of the CB-CB-S-CA dihedral angle in VTX in the independent simulations (left) and the resulting PDF (right). The dihedral angle is sampled every ns in the five binding sites (colored separately in blue, green, yellow, red, and purple). The ~40-degree (resp. -40-degree) angle region represents an upward (resp. downward) orientation of phenyl A.

Source data

Extended Data Fig. 5 Overlay of the mouse VTX-bound and antagonists-bound structures.

Overlay of the m5-HT3AR structure bound to VTX (colored as in Fig. 2) to different antagonist-bound structures (colored in gray) a, palonosetron (6Y1Z), b, palonosetron (6W1Y), c, ondansetron (6W1M) and d, alosetron (6W1J). Complementary subunits ECDs were used for superimposition. The ligands are shown as sticks (VTX colored in salmon and antagonists in red). The view is similar to that of Fig. 2e. The resemblance of the VTX-bound structure with the antagonist-bound structures extends to the organization of side chains neighboring the ligand on the complementary subunit side. This extremely conserved and rigid organization of the complementary side of the binding pocket appears to be a hallmark of the 5-HT3A receptor and is also observed in serotonin-bound structures representative of an active state. Diverse chemical structures thus fit in with only a little accommodation of the binding site moiety located on the complementary subunit. The largest differences among the VTX and setron-bound receptor structures are observed for the conformation of loop C and, more importantly, in the subunit-subunit interface reorganization12,17. In other words, the quaternary structure re-arrangement dominates over tertiary structure deviations. Still, the position of the backbone of loop C shows the biggest deviation among the VTX- and antagonist-bound structures, in line with the classical notion that it acts as a flexible lid on the orthosteric site of every pLGIC.

Extended Data Fig. 6 Comparison of VTX binding site residues and ligand-interactions in human and rodent receptors and mutational analysis of determinants for VTX activity.

a, Overview of the loop segments that form the orthosteric binding site in the 5-HT3A receptor (upper panel) and the 2D ligand-protein interaction diagram of vortioxetine with the h5-HT3AR (lower panel). Binding elements are indicated in colors: Loops A-C on the principal subunit and loops D-G on the complementary subunit. b, Multiple sequence alignment of the loop sequences in mouse (green), human (blue), rat (orange), and guinea pig (red) 5-HT3A receptors. Non-conserved positions are indicated with *. The presence and type of direct molecular interaction with VTX rings A, B, and C are indicated below the alignment: Hydrophobic = HP, hydrogen bond = HB, cation/pi=CP; pi/pi=PP, polar=PO. c,d, Amino acid sequence of loops D, G, F, and C in WT and mutant human, guinea pig, and rat 5-HT3A receptors with corresponding representative traces of the current response to the 3-step sequential protocol applying twice 10 μM 5-HT and 10 μM VTX in between that defines the receptor response-phenotype. To the right is a summary of the VTX phenotype for the constructs. Bar graphs represent the mean ± SEM (n = 7 independent oocyte recordings).

Source data

Extended Data Fig. 7 Image analysis workflow, h5-HT3AR in detergent, apo dataset.

a, Schematic of the image analysis workflow b, Example of one micrograph from the 7660 in the dataset.c, Selected 2D class averages corresponding to pentameric receptors. d, Side views of the reconstructions used for model building of the apo resting (left) and the apo active-distorted conformations (right). The density-modified map is colored according to the local resolution (FSC threshold of 0.5). e, Heat map of the angular distribution of particle projections for the reconstructions. f, Gold-standard FSC curves. The dotted line represents the 0.143 FSC threshold.

Extended Data Fig. 8 Structural comparisons of human apo resting 5-HT3A receptor.

a, Conservation-colored cartoon representation of a single subunit of the h5-HT3AR. Pink positions are conserved between mouse and human and positions in cyan are non-conserved b, Cartoon representation of the h5-HT3AR with a single subunit color-coded according to the RMSD calculated from pentameric superimposition with the structure of the palonosetron-bound mouse 5-HT3A receptor (PDB 6BE1). c, Close-up view of two opposing pore helices in an overlay of the apo (PDB 8BL8) and palonosetron-bound mouse 5-HT3A receptor structures and apo human 5-HT3A receptor structure. Pore lining residues are depicted as sticks. d,e, Zooms of loop C viewed from the top in the overlay of the apo and palonosetron-bound mouse 5-HT3A receptor structures with the apo human 5-HT3A receptor structure. The difference of loop C capping is small, and may merely reflect the difficulty of modeling this flexible zone rather than relevant species differences. e, Sequence differences could also explain the small difference in capping that might produce attractive interactions between the Met223-Glu224 motif on loop C and Lys195 on the complementary subunit in the human receptors, but repulsive interaction between the Ile201-Asp202 motif on loop C and Glu173 in the mouse receptor.

Extended Data Fig. 9 Image analysis workflow, h5-HT3AR in complex with VTX, in detergent or in nanodiscs dataset.

The top panels A to F correspond to the detergent dataset, the bottom panels G-L correspond to the nanodisc dataset. a,g, Schematic of the image analysis workflow b,h, Example of one micrograph from the dataset (size 1618 micrographs for the detergent dataset, 3290 for the nanodisc dataset). c,i, Selected 2D class averages of the final particles set. d,j, Side views of the post-processed reconstructions for the whole receptor (left) and the ECD (right). The maps are colored according to the local resolution. e,k, Heat map of the angular distribution of particle projections. f,l, Gold-standard FSC curves for the extracellular domain reconstruction. The dotted line represents the 0.143 FSC threshold.

Extended Data Fig. 10 Saturation binding curves for [3H]-GR65630 at WT h5-HT3AR and h5-HT3AR-V202I.

a,b, Specific binding (circles) and non-specific (squares) binding of [3H]GR65630 to membranes from HEK293 cells expressing WT (a) and V202I mutant (b) human 5-HT3AR is shown. The incubation time was 1 h at room temperature in 10 mM HEPES buffer, pH 7.4. Granisetron (1 µM) was used to define non-specific binding. Data points represent the mean ± SEM of 3 determinations. The full line is the fit of equation [RL] = [L] Bmax/([L] + Kd) where [RL] is the specific binding, [L] is the concentration of [3H]GR65630, Bmax is the maximum binding capacity, and Kd is the dissociation constant. The estimated Kd was 0.82 ± 0.06 nM and 0.62 ± 0.05 nM for WT and V202I, respectively.

Supplementary information

Supplementary information

Recovery from VTX inhibition with Supplementary Figs. 1–3; Structure–Activity Relationship of VTX analogs with Supplementary Figs. 4 and 5; Supplementary Tables 1–3.

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Supplementary Data 1

Numerical source data for Supplementary Information items.

Source data

Source Data Figs. 1, 4 and 5 and Extended Data Figs. 4 and 6

Numerical source data for all bar graphs.

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López-Sánchez, U., Munro, L.J., Ladefoged, L.K. et al. Structural determinants for activity of the antidepressant vortioxetine at human and rodent 5-HT3 receptors. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01282-x

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