Structure-based discovery of conformationally selective inhibitors of the serotonin transporter

Abstract

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Keywords: depression; docking; functional selectivity; serotonin transporter; ultra-large libraries.

Figure 1.. Docking-derived inhibitors of serotonin transporter.

(A) Inhibition of [³H]5-HT transport by docking-derived molecules, tested at 30 μM (Table S1). (B) Radio-ligand displacement of the top five docking hits (representative curves; summary data in Table S2). (C) 2D Chemical structures of the top five docking hits and their docked poses. Interactions are depicted as black dashed lines, ligand carbons in cyan and protein carbons in grey. Oxygens for both protein and ligand are red, nitrogen blue, and sulfur yellow. (D) Chemical structures of the parent compound, ‘2313 and its optimized analogs ‘8090 (Ki 14 nM) and ‘8219 (Ki 3 nM). The variable group in the analogs versus the parent are colored in magenta. Comparing the docked poses of (E) the parent ‘2313 (F) ‘8090 and (G) ‘8219. Dashed circles represent modeled improved stacking of F335 with ring substitutions in going from parent to the optimized analogs.

Figure 2

(A) Kinetics of inhibition by 0.15 μM ‘8219 and 0.7 μM ‘8090 (representative experiment). 5-HT transport into HeLa cells transfected with rSERT was non-competitively inhibited by both compounds. There was a small but statistically insignificant increase in KM with ‘8219 (0.69 ± 0.25 μM (SEM), n=6) and a decrease with ‘8090 (0.47 ± 0.05 μM, n=5) compared with uninhibited control (0.59 ± 0.18 μM, n=8). P values for paired t-tests were 0.99 and 0.18, respectively. Vmax was significantly decreased for both compounds from 1.7 ± 0.25 pmol/m/well for control (n=8) to 1.1 ± 0.2 pmol/m/well (n=6) for ‘8219 and 0.69 ± 0.1 pmol/m/well (n=5) for ‘8090. P values for paired t-tests were 0.018 and 0.011, respectively. (B) Efflux of accumulated [3H]5-HT induced by extracellular unlabeled 20 μM 5-HT, 10 μM ‘8219 or 10 μM ‘8090. 5-HT induced marked efflux of radiolabel, 8.3 ± 1.1 fmol/m (SEM)(n=6), relative to control (no addition) 0.63 ± 0.39 fmol/m (P=7×10–4 in 2-sample t-test, n=6). ‘8219 slightly increased efflux (1.8±0.6 fmol/m (SEM) but the increase was not significant (P=0.18, n=6) and ‘8090 barely increased efflux (0.12±0.09 fmol/m (SEM)(P=0.25, n=5). (C) Na+ and Cl− increased ‘8219 affinity in equilibrium displacement of [125I]β-CIT (representative experiment). Membranes from cells expressing SERT were incubated with 0.1 nM [125I]β-CIT and the indicated concentrations of ‘8219 in PBS/CM (control, blue line and circles), PBS/CM in which Na+ was replaced with NMDG+ (black line and circles) or Cl− was replaced with isethionate (red line and circles). The presence of Cl− increased ‘8219 inhibitory potency over 4-fold, from a KI of 21 ± 3 nM to 4.8 ± 1.0 nM (SEM) (n=4, P=0.001). Na+ increased ‘8219 inhibitory potency 131-fold, from a KI of 527 ± 143 nM to 4.0 ± 0.4 nM (SEM) (n=4, P=0.004).

Figure 3.. Influence of ‘8219, ‘8090 and ibogaine on SERT conformation.

(A) Effects on Cys277 reactivity in the cytoplasmic pathway. Rates of Cys277 modification in membranes from HeLa cells expressing SERT-S277C by MTSEA was measured by the decrease in β-CIT binding activity after treatment with MTSEA in the presence of the indicated compounds (cocaine, 10 µM, n=7; ibogaine, 10 µM, n=10; ‘8219, 0.05 µM, n=3; ‘8090 0.15 µM, n=4; imipramine, 0.6 µM, n=3; fluoxetine, 0.13 µM, n=3; citalopram, 0.03 µM, n=3; paroxetine, 0.02 µM, n=3). The reference compounds cocaine and ibogaine are shown at the top, and several clinically used antidepressants are shown at the bottom of the plot. Between them are ‘8219 and ‘8090. The control rate was 75 ± 15 s⁻¹M⁻¹ (n=18) and error bars represent SEM. (B) Effects on Cys277 reactivity in the absence of Na⁺ (replaced by NMDG). Rates were measured as above for control (n=6), ‘8219 (2 µM, n=6) and ‘8090 (2 µM, n=5). The control rate was 26 ± 5 s⁻¹M⁻¹ and the error bars represent SEM. ‘8219 significantly (P=0.04) increased the rate relative to control. (C) Effects on Cys107 reactivity in the extracellular pathway. Rates of Cys107 modification in HeLa cells expressing SERT-Y107C was measured by the decrease in residual transport activity after treatment with MTSET in the presence of the indicated compounds, each present at a saturating concentration (10x KD). Rates were measured as above for control (n=8), cocaine (n=3), ibogaine (n=4), ‘8219 (n=7) and ‘8090 (n=4). The control rate was 122 ± 14 s⁻¹M⁻¹. *=P<0.05 (paired t-test).

Figure 4.. Structural fidelity between the docked and cryo-EM poses of ‘8090.

(A) Cryo-EM density maps of ‘8090 bound to SERT in the outward-open state (PDB: 7TXT, EMBD: EMD-26160. (B) Comparison of ‘8090’s pose in cryo-EM structure (cyan) and the predicted docked pose in the inward open state of SERT (orange). (C) Comparison of ibogaine’s pose in cryo-EM determined inward open state structure (green, PDB ID: 6DZZ) and outward open state of structure of SERT (magenta, PDB ID: 6DZY). (D) Ligand cryo-EM determined pose (cyan) overlaid with the docked pose (yellow) in the outward open active site of SERT. (E) 2D outline of protein−ligand interactions between ‘8090 and SERT.

Figure 5.. Anti-depressive-like effects of compounds 8090 and 8219 in the tail suspension test with Vmat2 mice.

(A-B) Acute effects of ‘8090 (0.5, 1, and 2 mg/kg) and ‘8219 (0.1, 0.5, and 1 mg/kg) 30 min after injection in wild-type (WT) and VMAT2 heterozygous (HET) mice. Controls were given the vehicle (Veh), 20 mg/kg fluoxetine (Flx), or 30 mg/kg ibogaine (Ibo). (C-D) Persistent effects of ‘8090 and ‘8219 1 day post-injection in WT and VMAT2 HET mice. N=8–10 mice/genotype/treatment. All primary statistics are found in Table S9.

Figure 6.. The learned helplessness experimental design with sucrose preference and tail suspension using C57BL/6J mice.

(A) Top: Sub-chronic administration with the vehicle, 2 or 5 mg/kg ‘8090, or 5 mg/kg ‘8219 for 2 weeks followed immediately by chronic daily treatment throughout learned helplessness (LH) training. Bottom: Continued daily injections during the first 13 days of LH testing, followed by the withdrawal of treatment during testing. Abbreviations: Treat, treatment; Habit, habituation to the LH apparatus; LH, LH training; SE, shock escape testing; W-W, water-water pairing; S-W, sucrose-water pairing; IFS, intermittent foot-shock; FR, overnight food restriction; Strobe, strobe light during the dark cycle; Spray, the mouse and bedding were sprayed with water and the bedding was changed 2-days later; TS, tail suspension; EZM, elevated zero maze; RAM, radial 8-arm maze; OF, open field; and FSS, foot-shock sensitivity. (B) Sucrose preference in LH mice. N=10 mice/treatment. (C) Tail suspension in LH mice. N=10 mice/treatment. All primary statistics are in Table S9.

Figure 7:. The new SERT inhibitor ‘8219 reduces opioid withdrawal symptoms.

(A) Effect of systemic ‘8219 (10 mg/kg; N=9) on naloxone-precipitated morphine (M) withdrawal. (B) Effect of systemic paroxetine (PRX; 10 mg/kg; N=9) on withdrawal. Data are presented as means ± SEM with unpaired Student’s t-tests comparing effects of ‘8219 and paroxetine to saline and 20% ethanol, respectively.