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Photoaffinity‐Mediated Identification of a Third Citalopram Analog Binding Site on the Serotonin Transporter
Author(s) -
Tomlinson Michael J.,
Kumar Vivek,
Newman Amy H.,
Lever John R.,
Henry L. Keith,
Vaughan Roxanne A.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.680.1
Subject(s) - serotonin transporter , citalopram , serotonin plasma membrane transport proteins , pharmacophore , chemistry , transporter , serotonin , allosteric regulation , serotonin uptake inhibitors , 5 ht receptor , binding site , ligand (biochemistry) , pharmacology , biochemistry , receptor , fluoxetine , biology , gene
In the CNS, serotonin (5HT) controls anxiety and mood, and disorders caused by imbalanced 5HT include Major Depressive Disorder and Autism Spectrum Disorder. The serotonin transporter (SERT) is a major regulator of synaptic 5HT levels and is the target for antidepressants such as escitalopram (S‐CIT) and fluoxetine, which bind to and inhibit the transporter to elevate free transmitter. The efficacy of these drugs is moderate and many patients experience inadequate relief, indicating the need for better understanding of mechanisms or development of novel therapeutics. Antidepressant effects on SERT are complex, as many induce high‐affinity actions at the S1 orthosteric site, as well as allosteric modulation of S1 function through low‐affinity binding to a distinct S2 domain. To examine S‐CIT mechanisms we developed two novel photoaffinity ligands, [ 125 I]VK 03‐83 and [ 125 I]VK 03‐51, that possess identical S‐CIT pharmacophores but have cross‐linking moieties appended to different positions on the citalopram core. Our findings show that both ligands bind to SERT, inhibit 5HT transport, and allosterically regulate S1 function, but display distinct pharmacological characteristics that indicate differential binding mechanisms. [ 125 I]VK 03‐83 labeling of SERT is fully inhibited by 5HT and typical SERT inhibitors, with an S‐CIT K i of ~1 nM, consistent with binding to S1. In contrast, although [ 125 I]VK 03‐51 labeling is fully displaced by the homologous ligand, it is inhibited only ~30% by S‐CIT or other standard SERT inhibitors, and shows no displacement by 10 μM 5HT, indicative of binding to multiple sites. The S‐CIT‐displaceable portion of [ 125 I]VK 03‐51 labeling occurs with a K i of ~1 nM, consistent with binding at S1, whereas the fraction of labeling that is not sensitive to ≥10 μM concentrations of S‐CIT, other inhibitors, or 5HT likely does not occur at either S1 or S2, which should both be fully occupied by these conditions. These findings suggest that [ 125 I]VK 03‐51 interacts with SERT at a third site that has not been previously detected by reversible binding or co‐crystallization approaches, but is revealed by covalent cross‐linking. This site may function in transport inhibition or allosteric regulation, and suggests a domain for exploration of transporter mechanisms or medication development. Support or Funding Information NIH DA027845 and UNDSMHS Seed Grant (LKH and RAV), NIDA Intramural Research Program (AHN) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .