Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity

Abstract

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally^1-4. 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 derivatization^5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT_2A receptor (5-HT_2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT_2A or the 5-HT_2B receptors. Structure-based optimization led to the 5-HT_2AR 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-HT_2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT_2AR. 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-HT_2AR 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.

Extended Data Figure 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.

Extended Data Figure 2.. Pharmacological profiles of (S)-65, (R)-69, and (R)-70.

(A) Screening of (S)-65, (R)-69, and (R)-70 across the GPCRome (320 receptors) using the PRESTO-Tango platform 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 Figure 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 (2^nd panel), (R)-69 (3^rd panel) and (R)-70 (bottom panel) on the 5-HT_2AR. 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.

Extended Data Figure 4.. CryoEM workflow.

(A) Workflow of cryo electron microscopy (cryo-EM) data processing of the 5-HT_2AR/miniGα_q/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-HT_2AR, E, (R)-69 and F, α5 helix of miniGα_q/i.

Extended Data Figure 5:. Comparison between the cryo-EM structure of (R)-69 bound 5-HT2AR/miniGq/i and other 5-HT2AR structures.

(A) 5-HT_2AR - miniGα_q/i complex interactions. Arrow points to opening of TM5/6 in active (R)-69 bound 5-HT_2AR state. (R)-69 bound 5-HT_2AR in blue; miniGα_q/i in gold. Residues involved in hydrophobic interactions are labeled in grey while residues involved in H-bond interactions are labeled in red. (B) Ligand specific interactions with 5-HT_2AR. (R)-69 in magenta. LSD bound 5-HT_2AR structure in tan; LSD in orange. 25CN-NBOH bound 5-HT_2AR 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-HT_2AR ligand-binding pocket from the extracellular side; bottom: expansion of binding pocket of 5-HT_2AR 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 S159^3.36. In all panels the Ballesteros-Weinstein numbering is shown in superscript for each residue.

Extended Data Figure 6:. Pharmacokinetic analysis of (R)-69 and (R)-70.

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 Figure 7.. Null activity, startle reactivity, anti-depressant-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 Figure 8.. Spontaneous locomotion, conditioned place preference (CPP), and behavioral 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 analyzed 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) Behavioral 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 behavioral sensitization to cocaine increases across days 1–3 and remains high through testing. (R)-69-injected mice do not show behavioral 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 Figure 9.. Anxiety-like behaviors 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 traveled 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 Figure 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).

Figure 1.. Bespoke ultra-large virtual library approach.

(A) Drugs containing a tetrahydropyridine motif. (B) Three types of tetrahydropyridines (±)−1 to (±)−3 from commercially available alkynes 6 and 7, primary amines 4, and α,β-unsaturated carbonyl compounds 5. (C) Generation of a virtual library of 75 million tetrahydropyridines for docking against a homology model of the 5-HT_2AR.

Figure 2.. Large scale docking screen of a THP virtual library discovers new 5-HT₂R ligands.

(A) The initial 17 compounds synthesized and assayed; of these four had 5-HT_2AR or 5-HT_2BR activity (boxed). (B) Binding of the four actives at human 5-HT₂Rs in radioligand competition binding assays. Concentration-inhibition curves at 5-HT_2AR and 5-HT_2BR versus [³H]-LSD, and 5-HT_2CR versus [³H]- Mesulergine. [3H]-LSD, and [³H]-Mesulgergine concentrations of 0.5 nM, and 1.0 nM were used, respectively. K_D values of [³H]-LSD at human 5-HT_2AR and 5-HT_2BR were 0.33 and 0.91 nM, respectively, and K_D value of [³H]-mesulergine at human 5-HT_2CR was 0.67 nM^,. LSD (5-HT_2AR, 5-HT_2BR) and Ritanserin (5-HT_2CR) were used as positive controls. (C) Concentration-response curves of the four actives at stable 5-HT_2AR, 5-HT_2BR, and 5-HT_2CR-INI Flp-In293 cell lines in Ca²⁺ assays. Data in (B) and (C) represent the mean ±S.E.M from n=3 independent experiments. (D) Docked poses of characteristic molecules compared to that of LSD (left panel). In all panels, Ballesteros-Weinstein residue numbering is shown in superscript.

Figure 3.. Structure-guided discovery of 5-HT_2AR agonists and antagonists.

(A) Dose response competition binding assays of (S)-65, (R)-69, and (R)-70 against [³H]-LSD for 5-HT_2AR and 5-HT_2BR, and [³H]-Mesulergine for 5-HT_2CR. [³H]-LSD and [³H]-Mesulgergine concentrations of 0.3 nM and 1 nM were used, respectively. LSD (5-HT_2AR, 5-HT_2BR) and Risperidone (5-HT_2CR) were used as positive controls. (B) Concentration-response curves of (S)-65, (R)-69, and (R)-70 at stable 5-HT_2AR, 5-HT_2BR, and 5-HT_2CR-INI Flp-In293 cell lines in Ca²⁺ assays. (R)-69 and (R)-70 are strong partial G_q agonists, whereas (S)-65 acts as an antagonist. Data in (A) and (B) represent the mean ±S.E.M. from n=3 independent experiments. The pKi/EC50 values between 5-HT_2AR, 5-HT_2BR and 5-HT_2CR were compared using a one-way ANOVA with Dunnett’s post-test (* = p<0.05, ** = p<0.01, *** = p<0.001, and **** =p<0.0001). (C) 2D structures, binding and functional affinities for the new ligands across the 5-HT₂R subtypes. (D) Docking poses of (S)−65 (left panel), (S)−69 (the better scoring enantiomer; middle panel) and (R)-70 (right panel). 5-HT_2AR is shown in gray, the docked compounds are shown as capped sticks with carbons in yellow. In all panels the Ballesteros-Weinstein residue numbering is shown in superscript.

Figure 4.. Structure of 5-HT_2AR bound to (R)-69 by cryo-EM.

(A) Overall cryo-EM map (left) and model (right) of 5-HT_2AR bound to (R)-69 in complex with mini-G_q/i. (B) Schematic of ligand-specific interactions of (R)-69 with 5-HT_2AR orthosteric residues. A salt bridge with D155^3.32 is shown as a red dashed line. Color code for residues and interactions: green: hydrophobic, blue: polar, red: negatively charged, grey: glycine, green solid line: Pi-Pi stacking interaction. (C) Specific residues in the binding pocket that interact with (R)-69 are shown as sticks and labeled. (R)-69 is shown as magenta-colored sticks. A salt bridge with D155^3.32 is shown as a red dashed line. (D) Cryo-EM density for (R)-69. (E) Comparison of the computationally predicted and experimentally resolved binding poses of (S)-69 and (R)-69, respectively. The cryo-EM structure in magenta is superposed on (S)-69 docked to the 5-HT_2AR homology model in green. Ballesteros-Weinstein residue numbering is in superscript in panels (C) and (E).

Figure 5.. Head twitch, PPI responses, and anti-depressant-like actions of (R)-69 and (R)-70 in mice.

(A) Head twitch responses (HTRs) in C57BL/6J mice during the first 30 min after injection (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. HTRs are low in (R)-69 and (R)-70 groups and they partially block the LSD effects. (B) PPI in C57BL/6J mice treated (i.p.) with vehicle, 1 or 3 mg/Kg (R)-69, 1 or 3 mg/Kg (R)-70, or LSD. PPI is unaffected with (R)-69 or (R)-70 relative to vehicle and LSD disruption. (C-D) Immobility in tail suspension at 30 min and 24 h with WT and VMAT2 heterozygous (HET) mice after a single injection (i.p.) of vehicle, 20 mg/Kg fluoxetine (FLX), 0.5 or 1 mg/Kg (R)-69, or 0.5 or 1 mg/Kg (R)-70. Acute genotype differences are seen with vehicle and 0.5 mg/Kg (R)-70; at 24 h effects are present with vehicle, FLX, and 0.5 mg/Kg (R)-70. In WT mice immobility is enhanced with acute administration of FLX relative to vehicle and at 24 h it is increased with FLX compared to all other groups. In VMAT2 HETs, 30-min after administration FLX, 1 mg/Kg (R)-69, and 0.5 and 1 mg/Kg (R)-70 reduce immobility and at 24 h 0.5 and 1 mg/Kg (R)-69 and (R)-70 are efficacious compared to vehicle. Results presented as mean ±s.e.m. in the figure, Ns are provided in Methods section. Primary statistics are found in Supplementary Table 4. In this figure the Bonferroni pair-wise corrected p-values (ps) across multiple comparisons for LSD, vehicle, or FLX are depicted for the value closest to p<0.05 (panels A-D) or within a single comparison (p) between or within genotypes (panels C-D).

Figure 6.. Experimental design, sucrose preference, and tail suspension for the learned helplessness experiment.

(A) Experimental design. (B) Sucrose preference in C57BL/6 mice assigned to the non-foot-shock (NFS) and foot-shock (FS) conditions and treated (i.p.) with vehicle, 1 mg/Kg (R)-70, 1 mg/Kg psilocin, or 10 mg/Kg ketamine. During the initial sucrose-water (S-W) pairing, mice in the FS condition have a lower preference for sucrose than the NFS mice. Among the NFS animals, no treatment effects are found within days. By comparison among FS mice, sucrose preference is reduced in the vehicle controls compared to the (R)-70 and psilocin groups on days 0, 1, and 3. The effects of ketamine are delayed but become manifest on days 1 and 3. (C) Tail suspension testing in the same mice. Times of immobility are low and stable across time in the NFS mice. In the FS condition, 30-min following treatment (day 0), immobility is high in the vehicle control relative to the (R)-70 group and this effect persists through day 14. Relative to the FS vehicle control, psilocin and ketamine show efficacy on days 1 and 4, with the effects of psilocin lasting to day 9 post-injection. 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 vehicle showing the value closest to p<0.05 within the FS condition or within a single comparison (p) from the vehicle (panels B-C) or condition (panel B).