(S)- and (R)-fluoxetine as native markers in mass spectrometry (MS) binding assays addressing the serotonin transporter

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 nM and 5.2±0.9 nM 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.