title: Antidepressants targeting the serotonin reuptake transporter act via a competitive mechanism.
abstract: Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099-3108, 1973). Two different SERT-selective radioligands, [(3)H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [(3)H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [(3)H]DASB and [(3)H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC(50)) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-beta-carboxy-3-beta-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.
authors.affiliation: Department of Biochemical Pharmacology, Roche Pharmaceuticals, Palo Alto, CA 94304, USA. subbu.apparsundaram@roche.com
authors.forename: Subbu
authors.initials: S
authors.lastname: Apparsundaram
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Stockdale
authors.forename: Robert A
authors.initials: RA
authors.lastname: Henningsen
authors.forename: Marcos E
authors.initials: ME
authors.lastname: Milla
authors.forename: Renee S
authors.initials: RS
authors.lastname: Martin
mesh_terms: D000818:Animals; D000928:Antidepressive Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D001667:Binding, Competitive; D002462:Cell Membrane; D002540:Cerebral Cortex; D006801:Humans; D011869:Radioligand Assay; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins / Q000187:drug effects*; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology
chemical_list: D000928:Antidepressive Agents; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors