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

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.