There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1,2,3,4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines—a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C–H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5,6,7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.
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The cryo-EM density map and corresponding coordinates for 5-HT2AR in complex with the new docking hit (R)-69 have been deposited at the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB), respectively, under the following accession codes: EMD-24378 and 7RAN. The full THP library of compounds may be freely accessed online (http://thp.docking.org/). All active compounds are available from the authors on request. Sequences used to generate the 5-HT2AR homology model are available at UniProt (P28223 and 5P41595) or from the PDB (4IB4 (chain A), 4NC3 (chain A), 5TVN (chain A)). Figures with associated raw data include Figs. 2 and 3 (the underlying activities are uploaded in the Source Data); Fig. 4 and Extended Data Figs. 4 and 5 (electron density maps and associated files are deposited at the PDB); Fig. 5 (underlying numbers are uploaded in an Excel file); Extended Data Figs. 2, 3 and 6 (underlying activities are uploaded in the Source Data); Extended Data Table 3 (underlying numbers are supplied in Supplementary Table 5); and Supplementary Fig. 3 (underlying numbers are uploaded in an Excel file). Further underlying data are provided in Extended Data Table 4 (cryo-EM data collection, refinement and validation); Supplementary Data 1 (synthesis procedures for active compounds, chemical purity of active ligands, spectra and 3D crystallography structures); and Figs. 5 and 6. Extended Data Figures 7–10 depict the raw data points with mean ± s.e.m. (Prism files; GraphPad Software) and Supplementary Table 4 displays the statistics from behavioural data (IBM SPSS, v.27 and v.28). All raw behavioural data are uploaded in Excel files. Source data are provided with this paper.
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We thank the staff at the Schrödinger for donating its Maestro and FEP+ packages and programs and OpenEye Scientific for donating the Omega and OEChem programs; E. Montabana for support with data collection; M. Huffstickler, C. Ritter and C. Means for helping with the behavioural testing; and J. Zhou for breeding, genotyping and maintaining the VMAT2 mice; and the staff at the NIDA Drug Supply Program for providing us with (+)-LSD-(+)-tartrate and psilocin. This work was supported by DARPA HR001119S0092 (to B.L.R., G.S., W.C.W. and B.K.S.) and by NIH grants R35GM122473 (to J.A.E.), R35GM122481 (to B.K.S.), R37DA045657 (to B.L.R.), R01MH11205 (B.L.R. and B.K.S.) and GM71896 (to J.J.I.). Some of the behavioural experiments were conducted with equipment and software purchased with a North Carolina Biotechnology Center grant. The views, opinions and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views, policies or endorsement of the Department of Defense or the US Government.
J.A.E., D.N.C., O.S.K., B.L.R., K.K., B.K.S., A.L.K. and J.J.I. have filed a patent (W02022067165) around the new THP agonists through their universities. B.K.S. is a founder of Epiodyne and, with J.J.I., BlueDolphin. B.K.S., J.J.I. and G.S. are co-founders of Deep Apple. B.L.R. is a scientific founder of Onsero Therapeutics. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 (a) Synthetic routes to access densely functionalized, diastereoselective tetrahydropyridines 1 to 3.
Building blocks used for tetrahydropyridines (±)-1 and (±)-2: R1, R5, R6 = alkyl, aryl, heteroaryl; R2 = H, alkyl; R3, R4 = H, alkyl, aryl, heteroaryl. Building blocks used for tetrahydropyridines (±)-3: R1 = alkyl; R5 = alkyl, aryl, heteroaryl; R2 = H, alkyl; R3, R4 = H, alkyl, aryl, heteroaryl. See Supplementary Data 1 for detailed reaction conditions and procedures. (b) Alternate routes for installation of R1 substituent. See Supplementary Data 1 for detailed reaction conditions and procedures.
(a) Screening of (S)-65, (R)-69, and (R)-70 across the GPCRome (320 receptors) using the PRESTO-Tango platform10 with agonists present at 3 μM concentrations; for viewability, only 1-in-4 receptors are listed on the x-axis. (b) Analysis of the function of (S)-65, (R)-69, and (R)-70 on various receptors shows the diverse profiles for each receptor. (c)-(f) Binding profiles of (S)-65, (R)-69, and (R)-70 on (c) hERG; human ether-a-go-go-related gene, (d) NET; norepinephrine transporter, (e) DAT; dopamine transporter, and (f) SERT; serotonin transporter. Data in (a), (b), (c), (d), (e) and (f) represent the mean ±S.E.M from n = 3 independent experiments.
Extended Data Fig. 3 Characterization and time course activities of functional bias of (R)-69 and (R)-70 at the human 5-HT2AR.
(a) BRET (Gq dissociation and β-Arrestin 2 association) activities of 5-HT (top panel), psilocin (2nd panel), (R)-69 (3rd panel) and (R)-70 (bottom panel) on the 5-HT2AR. Data represent the mean ± SEM from n = 3 independent experiments, each performed in triplicate. (b) Transduction coefficients for Gq and βArr2 at various time points. Transduction coefficients were measured (see METHODS) for each time point in (a) and then plotted vs time.
(a) Workflow of cryo electron microscopy (cryo-EM) data processing of the 5-HT2AR/miniGαq/i complex bound to (R)-69. (b) Local resolution estimation heat map and gold standard Fourier shell correlation (FSC) curve. Dashed line represents the overall nominal resolution at 0.143 FSC of 3.38 Å calculated using Phenix Mtriage and 3.45 Å calculated by Relion 3.1 after post-processing. (c) Angular distribution heat map of particles for cryo-EM reconstruction. (d) Cryo-EM density for TM1-7 of 5-HT2AR, E, (R)-69 and F, α5 helix of miniGαq/i.
Extended Data Fig. 5 Comparison between the cryo-EM structure of (R)-69 bound 5-HT2AR/miniGq/i and other 5-HT2AR structures.
(a) 5-HT2AR - miniGαq/i complex interactions. Arrow points to opening of TM5/6 in active (R)-69 bound 5-HT2AR state. (R)-69 bound 5-HT2AR in blue; miniGαq/i in gold. Residues involved in hydrophobic interactions are labelled in grey while residues involved in H-bond interactions are labelled in red. (b) Ligand specific interactions with 5-HT2AR. (R)-69 in magenta. LSD bound 5-HT2AR structure in tan; LSD in orange. 25CN-NBOH bound 5-HT2AR structure in light cyan; 25CN-NBOH in cyan. Arrow points to extension of (R)-69 and 25CN-NBOH towards TM5. (c) Top: view of the 5-HT2AR ligand-binding pocket from the extracellular side; bottom: expansion of binding pocket of 5-HT2AR bound to (R)-69 towards the cytosolic side of the receptor. (d) Proposed optimization of (R)-69 to engage in a hydrogen-bond interaction with S1593.36. In all panels the Ballesteros-Weinstein numbering is shown in superscript for each residue.
Concentration-time curves for (R)-69 (a) and (R)-70 (b) in male C57BL/6N mice following IP dosing of either 1 or 10 mg/Kg of the compounds. (c) Selected pharmacokinetic parameters for (R)-69 and (R)-70 in male C57BL/6N mice.
Extended Data Fig. 7 Null activity, startle reactivity, antidepressant-like actions of MDL 100907 and SB 242084, and total volume of sucrose and water consumed.
(a) Null activity during PPI for C57BL/6J mice treated (i.p.) with vehicle, 1 or 3 mg/Kg (R)-69, 1 or 3 mg/Kg (R)-70, or 0.3 mg/Kg LSD. Null activity increases with 1 mg/Kg (R)-69 group. (b) Startle reactivity during PPI with C57BL/6J mice undergoing the same treatments. No significant effects are found. (c) Immobility in tail suspension at 30 min and 24 h with WT and VMAT2 HET mice after a single injection (i.p.) of vehicle, 0.5 mg/Kg MDL 100907, or 1 mg/Kg SB 242084. Genotype differences are observed in acute and 24 h tests with vehicle and SB. In WT mice, immobility is increased acutely with MDL compared to vehicle and SB. The difference between the MDL and vehicle groups persists at 24 h. In VMAT2 HETs, immobility times in vehicle controls are high acutely relative to SB, with a trend (p = 0.055) for MDL. At 24 h, immobility times for vehicle-treated mice are higher than for MDL. (e) Total fluid consumed during the sucrose preference test with non-foot-shocked (NFS) and foot-shocked (FS) C57BL/6J mice given (i.p.) vehicle, 1 mg/Kg (R)-70, 1 mg/Kg psilocin, or 10 mg/Kg ketamine. FS mice drink more than NFS animals during water-water (W-W) and sucrose-water (S-W) pre-test pairings, as well as on post-injection days 0, 1, and 3. Results presented as mean ±s.e.m., Ns are found in Methods. Primary statistics are in Supplementary Table 4. In the figure the Bonferroni pair-wise corrected p-values (ps) across multiple comparisons for 1 mg/kg R)-69 or MDL showing the value closest to p < 0.05 (respective panels A, C) or within a single comparison (p) from the vehicle, MDL, or SB groups (panels C-D), or condition (panel E).
Extended Data Fig. 8 Spontaneous locomotion, conditioned place preference (CPP), and behavioural sensitization to (R)-69 and (R)-70.
(a) Baseline (30 min) and post-injection locomotion (30 min) in C57BL/6J mice after administration (i.p.) of vehicle, 1 or 3 mg/Kg (R)-69, 1 or 3 mg/Kg (R)-70, 0.3 mg/Kg LSD, 1 or 3 mg/Kg (R)-69 + 0.3 mg/Kg LSD, or 3 mg/Kg (R)-70 + 0.3 mg/Kg LSD. Since baseline activities are different among groups, the data are analysed as percent baseline in the next panel. (b) Percent change from baseline from mice in the same experiment. Compared to vehicle, different doses of (R)-69 or (R)-70 have neither stimulatory nor inhibitory effects on locomotion. The compounds blocked LSD-stimulated hyperlocomotion. (c) CPP in C57BL/6J mice to 3 mg/Kg (R)-69, 3 mg/Kg (R)-70, or 20 mg/Kg cocaine (i.p.). No group differences are present at acclimatization, while CPP is evident only in the cocaine group. (d) Behavioural sensitization across 5 consecutive days with a challenge on day 11 using C57BL/6J mice treated (i.p.) with 3 mg/Kg (R)-69 or 20 mg/Kg cocaine. While baseline activities (1 h) are similar between (R)-69- and cocaine-treated mice on day 1, baseline locomotion in the latter group is increased across days 2–5 and at challenge on day 11. Locomotor activities (2 h) are low across all post-injection days for (R)-69 mice, whereby behavioural sensitization to cocaine increases across days 1–3 and remains high through testing. (R)-69-injected mice do not show behavioural sensitization. Results presented as mean ±s.e.m. in the figure, Ns are found in Methods. Primary statistics are in Supplementary Table 4. In the figure the Bonferroni pair-wise corrected p-values (ps) across multiple comparisons to LSD, LSD + (R)-69, or LSD + (R)-70 showing the value closest to p < 0.05 (panel B) or within a single comparison (p) from cocaine on specific days (panels C-D)
Extended Data Fig. 9 Anxiety-like behaviours in non-foot-shocked (NFS) and foot-shocked (FS) learned helplessness mice.
(a) Percent time in the open areas in the elevated zero maze with C57BL/6J mice treated (i.p.) with the vehicle, 3 mg/Kg (R)-70, 1 mg/Kg psilocin, or 10 mg/Kg ketamine. Animals are tested 13 days post-injection. Mice in the FS condition spend less time in the open areas than NFS animals. Vehicle-treated FS mice spend less time in the open areas than the (R)-70, psilocin, or ketamine FS animals. (b) Latencies to enter the open areas of the maze. Latencies to enter the open areas are prolonged in the FS mice. (c) Distance travelled in the maze. FS mice ambulate within the maze over shorter distances than the NSF animals. Results presented as mean ±s.e.m. in the figure, Ns are provided in the Methods section. Primary statistics are found in Supplementary Table 4. In the figure the Bonferroni pair-wise corrected p-values (ps) across multiple comparisons for vehicle showing the treatment value closest to p < 0.05 (panel A) or within a single comparison (p) from condition (panels A-C).
Extended Data Fig. 10 Numbers of escapes, latency to escape, and foot-shock reactivity in non-foot-shocked (NFS) and foot-shocked (FS) learned helplessness mice.
(a) Number of escapes from foot-shock in C57BL/6J mice treated (i.p.) with the vehicle, 1 mg/Kg (R)-70, 1 mg/Kg psilocin, or 10 mg/Kg ketamine. All mice assigned to the FS condition have fewer escapes from foot-shock than the NFS animals. (b) Latency to escape from foot-shock in the same experiment. All mice in the FS condition have prolonged escape latencies compared to NFS animals. (c) Reactivity to foot-shock in the same experiment. No significant differences in response to foot-shock are detected among treatment groups or between mice in the NFS or FS conditions. All mice exposed to foot-shock (i.e., 0.1-0.3 mAmp) respond at a similar magnitude and this is higher than in the absence of foot-shock (0 mAmp). Results presented as mean ±s.e.m. in the figure, Ns are provided in the Methods section. Primary statistics are found in Supplementary Table 4. In the figure the Bonferroni pair-wise corrected p-values (ps) across multiple comparisons to 0 mAmp showing the intensity closest to p < 0.05 (panel C) or within a single comparison (p) from condition (panels A-B).
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Kaplan, A.L., Confair, D.N., Kim, K. et al. Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity. Nature 610, 582–591 (2022). https://doi.org/10.1038/s41586-022-05258-z