Setup and Evaluation of the TRACT Assay Principle for the In Vitro Functional Characterization of Modulators of the Serotonin Transporter and the Serotonin 2A Receptor

The human serotonin transporter (SERT) is a pertinent target for many psychiatric therapeutics and recreational drugs including new psychoactive substances. Current techniques to assess SERT inhibition typically monitor the uptake of radiolabeled or fluorescent substrates but face limitations regarding the use of nonendogenous substrates and/or low throughput. Inspired by a previously reported principle, coined 'TRACT' or 'transporter activity through receptor activation', a new bioassay to measure SERT inhibition was developed. The assay principle relies on SERT-mediated uptake of supplied serotonin (5-HT), precluding 5-HT-induced activation of a coexpressed modified serotonin 2A receptor (5-HT2AR). Upon SERT inhibition, more extracellular 5-HT is available for 5-HT2AR-activation, which is monitored via β-arrestin 2 recruitment, using functional complementation of a split-nanoluciferase and a luminescent read-out. Following optimization, the assay was successfully applied to three classes of antidepressants and the designer cathinone naphyrone. Obtained inhibition potencies ranged from 2.61 nM for paroxetine to 348 nM for naphyrone, and good agreement in rank order was obtained when compared with the outcome of a fluorescence-based and a radioactivity-based uptake assay. Our findings demonstrate that the TRACT assay principle is translatable to SERT, allowing the assessment of SERT inhibition via 5-HT2AR activation. Additionally, for amitriptyline and amoxapine, the TRACT assay indicated direct (non-SERT mediated) 5-HT2AR-modulating effects, caused by inverse agonism, as confirmed by the 5-HT2AR βarr2 recruitment assay. We also demonstrate a proof-of-principle for applying this system to detect SERT-mediated efflux. Overall, our findings support the utility of the TRACT assay for the characterization of SERT ligands.