Dynamic extracellular vestibule of human SERT: unveiling druggable potential with novel 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 discovery of a series of high-affinity allosteric inhibitors of hSERT in a novel 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 the serotonin transport in a non-competitive 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 a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose is validated by a site-directed mutagenesis study, 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.

Allosteric modulation of serotonin and dopamine transporters: New insights from computations and experiments

“…The classification and criteria of these two distinct subsites concurs with the binding poses of the lead compound Lu AF88273 for SERT allosteric inhibitors that were recently proposed ( Salomon et al, 2023 ). The lead compound Lu AF88273 was shown, through docking, MD simulations as well as mutagenesis experiments, to adopt two distinct binding poses IPC (S2–I) and CPI (S2-II) at the S2 site with high affinity, where the F556 residue would accommodate the binding with different dihedral angles similarly to what is observed in Fig.…”

“…These earlier studies have now led to the identification of compounds with therapeutic potential demonstrating similar allosteric effects on ligand dissociation but with significantly higher affinities for the allosteric site over the orthosteric site ( Plenge et al, 2020 ; Salomon et al, 2023 ). In addition to the observations on allosterically modulating ligand dissociation from the orthosteric site, allosteric effects have been observed in studies of MATs including effects on the affinity of ligands that bind S1, non-competitive inhibition of substrate translocation, and effects on behaviors in animal models ( Niello et al, 2020 ).…”

Alchemical Free Energy Calculations on Membrane-Associated Proteins

“…Finally, both absolute and relative FEP calculations were applied to the same target to reveal the binding pose of a known allosteric inhibitor and the SAR of its analogues to the same binding site. 189 ■ DISCUSSION…”

“…The alchemical perturbation from F- to Br- is energetically favorable in both ABC (ΔΔ G = −1.63 ± 0.39 kcal/mol) and ACB (ΔΔ G = −1.08 ± 0.57 kcal/mol) poses of the SERT likely due to favorable enthalpic contributions between the Br- and the negatively charged oxygens of nearby residues. Finally, both absolute and relative FEP calculations were applied to the same target to reveal the binding pose of a known allosteric inhibitor and the SAR of its analogues to the same binding site …”