TY - JOUR

T1 - Dynamic extracellular vestibule of human SERT

T2 - Unveiling druggable potential with high-affinity allosteric inhibitors

AU - Salomon, Kristine

AU - Abramyan, Ara M.

AU - Plenge, Per

AU - Wang, Lingle

AU - Bundgaard, Christoffer

AU - Bang-Andersen, Benny

AU - Loland, Claus J.

AU - Shi, Lei

N1 - Publisher Copyright: Copyright © 2023 the Author(s).

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - allosteric binding site

KW - allosteric modulator

KW - free energy perturbation

KW - molecular dynamics

KW - serotonin transporter

U2 - 10.1073/pnas.2304089120

DO - 10.1073/pnas.2304089120

M3 - Journal article

C2 - 37792512

AN - SCOPUS:85174317559

VL - 120

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 41

M1 - e2304089120

ER -