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( S )‐ and ( R )‐Fluoxetine as Native Markers in Mass Spectrometry (MS) Binding Assays Addressing the Serotonin Transporter
Author(s) -
Hess Marielle,
Höfner Georg,
Wanner Klaus T.
Publication year - 2011
Publication title -
chemmedchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.817
H-Index - 100
eISSN - 1860-7187
pISSN - 1860-7179
DOI - 10.1002/cmdc.201100251
Subject(s) - fluoxetine , chemistry , dissociation constant , serotonin transporter , enantiomer , serotonin plasma membrane transport proteins , radioligand , serotonin , pharmacology , stereochemistry , chromatography , binding site , biochemistry , receptor , biology
Abstract A recently established and validated LC–ESI‐MS/MS method for quantification of fluoxetine was used to implement MS binding assays for the human serotonin transporter (hSERT)—the primary target in the treatment of depression and emotional disorders. As a label‐free screening technique, MS binding assays offer the opportunity to perform kinetic, saturation and competition assays using both ( S )‐ and ( R )‐fluoxetine as native markers. In kinetic experiments, an association rate constant ( k +1 ) of 0.92±0.17×10 6 M −1 s −1 and a dissociation rate constant ( k −1 ) of 0.0032±0.0002 s −1 for ( S )‐fluoxetine binding to hSERT were determined. Saturation experiments provided K d values of 4.4±0.4 n M and 5.2±0.9 n M for ( S )‐ and ( R )‐fluoxetine, respectively; statistical analysis revealed that the two enantiomers are equipotent. In competitive experiments with ( S )‐fluoxetine as a marker, K i values were obtained for various known inhibitors with a broad range of affinities for hSERT that correlate well with literature data obtained from radioligand binding experiments with [ 3 H]imipramine. Additional competitive experiments using ( R )‐fluoxetine as a marker led to K i values for SERT inhibitors that deviate only marginally from those determined using the ( S )‐enantiomer. No changes in the rank order of affinities occurred, indicating that there is no difference in the binding characteristics of the two enantiomers.