Dynamic extracellular vestibule of human SERT: Unveiling druggable potential with high-affinity allosteric inhibitors

Abstract

The serotonin transporter (SERT) tightly regulates synaptic serotonin levels and has been the primary target of antidepressants. Binding of inhibitors to the allosteric site of human SERT (hSERT) impedes the dissociation of antidepressants bound at the central site and may enhance the efficacy of such antidepressants to potentially reduce their dosage and side effects. Here, we report the identification of a series of high-affinity allosteric inhibitors of hSERT in a unique scaffold, with the lead compound, Lu AF88273 (3-(1-(2-(1H-indol-3-yl)ethyl)piperidin-4-yl)-6-chloro-1H-indole), having 2.1 nM allosteric potency in inhibiting imipramine dissociation. In addition, we find that Lu AF88273 also inhibits serotonin transport in a noncompetitive manner. The binding pose of Lu AF88273 in the allosteric site of hSERT is determined with extensive molecular dynamics simulations and rigorous absolute binding free energy perturbation (FEP) calculations, which show that a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose are validated by site-directed mutagenesis and can explain much of the structure-activity relationship of these inhibitors using the relative binding FEP calculations. Together, our findings provide a promising lead compound and the structural basis for the development of allosteric drugs targeting hSERT. Further, they demonstrate that the divergent allosteric sites of neurotransmitter transporters can be selectively targeted.

Keywords: allosteric binding site; allosteric modulator; free energy perturbation; molecular dynamics; serotonin transporter.

Fig. 1.

Allosteric inhibition of [³H]imipramine ([³H]IMI) dissociation by Lu AF88273 and analogs. Data plotted as the dissociation rate of [³H]IMI in the presence of the indicated compounds (k_cpd) relative to no compound added (k_ctrl). Lu AF88273 inhibits the dissociation of [³H]IMI with an IC₅₀ of 2.1 [1.5; 3.0] nM (mean [SEM interval]). See Table 1 for allosteric potencies for all compounds. All experiments were performed on membranes of COS7 cells transiently transfected with hSERT WT. Data are shown as mean ± SEM, n = 3 to 4.

Fig. 2.

Effect of Lu AF88273 on [³H]5-HT transport. (A) Lu AF88273 inhibits [³H]5-HT uptake (black) with a K_i of 53 [38;74] nM (mean [SEM interval]), which is noticeably more potent than 5-HT (green). Data are shown as means ± SEM (error bars), n = 3. (B) Effect of indicated Lu AF88273 concentrations on [³H]5-HT transport. The K_m for [³H]5-HT in buffer only (ctrl) is not significantly different from the K_m values in the presence of Lu AF88273 (P > 0.05). The V_max of [³H]5-HT uptake decreases with increasing concentrations of Lu AF88273 but reached a plateau at 400 nM of Lu AF88273 (SI Appendix, Table S2), in accordance with a noncompetitive inhibition. For statistical analysis, the one-way ANOVA with the Dunnett multiple comparisons test was used. Data are shown as mean ± SEM (error bars), n = 3 to 5. See SI Appendix, Table S2 for K_m and V_max values. All experiments are performed in triplicates on COS7 cells transiently transfected with hSERT WT.

Fig. 3.

Lu AF88273 in its IPC pose is stably bound in the S2 site of hSERT in the presence of S1-bound imipramine. (A) An overview of hSERT immersed in the lipid bilayer, with the locations of the central S1 and extracellular S2 binding sites indicated. Imipramine bound in the S1 site and Lu AF88273 bound in the S2 site are in surface representations. (B and C) The representative ensembles of Lu AF88273 bound in both the CPI (B) and IPC (C) poses from their corresponding MD simulations. The Na⁺ and Cl⁻ ions bound in or near the S1 site are shown in yellow and green spheres, respectively. Note that F556 is flexible in the CPI pose, while it stacks with 6-Cl-indole moiety of Lu AF88273 in the IPC pose.

Fig. 4.

The IPC pose occupies the cavities that the CPI pose does not. Zoom-in views show distinct interactions between hSERT and Lu AF88273 in its CPI (A) and IPC (B) poses. Black dotted circles indicate the locations of the cavities at the TM1 and TM6 interface (formed by residues R104, Q332, F335, and S336) and at the TM11 and TM12 interface (dynamically formed after the rotation of F556). Black dashed lines indicate the interactions uniquely formed in the IPC pose.

Fig. 5.

Effect of hSERT point mutations on Lu AF88273 allosteric potency. Inhibition of the dissociation of [³H]IMI from various hSERT constructs with the indicated mutations. All mutations decrease the allosteric potency of Lu AF88273 relative to hSERT WT (dotted line). Data plotted as the [³H]IMI dissociation rate in the presence of Lu AF88273 (k_{Lu AF88273}) relative to no compound added (k_ctrl). See allosteric potencies for all mutants in SI Appendix, Table S5. The concentration of [³H]IMI was 12.3 nM. All experiments were performed on membranes of COS7 cells transiently transfected with the respective hSERT mutants. Data are shown as mean ± SEM, n = 3 to 4.