Dynamic extracellular vestibule of human SERT: Unveiling druggable potential with 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 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.

Series of (([1,1'-Biphenyl]-2-yl)methyl)sulfinylalkyl Alicyclic Amines as Novel and High Affinity Atypical Dopamine Transporter Inhibitors with Reduced hERG Activity.

Currently, there are no FDA-approved medications for the treatment of psychostimulant use disorders (PSUD). We have previously discovered "atypical" dopamine transporter (DAT) inhibitors that do not display psychostimulant-like behaviors and may be useful as medications to treat PSUD. Lead candidates (e.g., JJC8-091, 1) have shown promising in vivo profiles in rodents; however, reducing hERG (human ether-à-go-go-related gene) activity, a predictor of cardiotoxicity, has remained a challenge. Herein, a series of 30 (([1,1'-biphenyl]-2-yl)methyl)sulfinylalkyl alicyclic amines was synthesized and evaluated for DAT and serotonin transporter (SERT) binding affinities. A subset of analogues was tested for hERG activity, and the IC50 values were compared to those predicted by our hERG QSAR models, which showed robust predictive power. Multiparameter optimization scores (MPO > 3) indicated central nervous system (CNS) penetrability. Finally, comparison of affinities in human DAT and its Y156F and Y335A mutants suggested that several compounds prefer an inward facing conformation indicating an atypical DAT inhibitor profile.

Modifications to 1-(4-(2-Bis(4-fluorophenyl)methyl)sulfinyl)alkyl Alicyclic Amines That Improve Metabolic Stability and Retain an Atypical DAT Inhibitor Profile.

Atypical dopamine transporter (DAT) inhibitors have shown therapeutic potential in the preclinical models of psychostimulant use disorders (PSUD). In rats, 1-(4-(2-((bis(4-fluorophenyl)methyl)sulfinyl)ethyl)-piperazin-1-yl)-propan-2-ol (JJC8-091, 3b) was effective in reducing the reinforcing effects of both cocaine and methamphetamine but did not exhibit psychostimulant behaviors itself. Improvements in DAT affinity and metabolic stability were desirable for discovering pipeline drug candidates. Thus, a series of 1-(4-(2-bis(4-fluorophenyl)methyl)sulfinyl)alkyl alicyclic amines were synthesized and evaluated for binding affinities at DAT and the serotonin transporter (SERT). Replacement of the piperazine with either a homopiperazine or a piperidine ring system was well tolerated at DAT (Ki range = 3-382 nM). However, only the piperidine analogues (20a-d) showed improved metabolic stability in rat liver microsomes as compared to the previously reported analogues. Compounds 12b and 20a appeared to retain an atypical DAT inhibitor profile, based on negligible locomotor activity in mice and molecular modeling that predicts binding to an inward-facing conformation of DAT.

The antidepressant drug vilazodone is an allosteric inhibitor of the serotonin transporter.

Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [3H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.