(S)‐ and (R)‐Fluoxetine as Native Markers in Mass Spectrometry (MS) Binding Assays Addressing the Serotonin Transporter

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 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.

MS Binding Assays for D1and D5Dopamine Receptors

“…To enable quantification of bound SCH23390 even at concentrations <10 p m (LLOQ) in the binding samples, we tried to benefit from a concentration step in which the volume of the final sample containing the liberated, formerly bound marker is lower than that of the original incubation sample containing the target–marker complexes, as was also recently realized in SERT and NET MS Binding Assays –. Thus, incubation was performed in deep‐well plates in a total volume of 1000 μL per well.…”

Low physical fitness is a strong predictor of health problems among young men: a follow-up study of 1411 male conscripts

“…A conscript's physical fitness index (CPFI) was calculated using the following formula: (12-min running test result [metres] + 100 × Muscle fitness test points)/200. The formula is based on standard practice in the Finnish Defence Forces since 1982 [21]. Because excellent results in the Cooper's test were uncommon (< 4%), the two highest levels, good and excellent, were combined to obtain a group of equal size for comparison between different fitness categories.…”

Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters

“…This is typically achieved by denaturation of the target protein and subsequent elution of the formerly bound marker by an organic solvent. According to this strategy MS Binding Assays for several neurotransmitter receptors and transporters have been established over the past few years …”

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