MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)‐Indatraline as Native Marker

We herein present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis‐configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)‐indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)‐indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)‐indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT.