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<p>Synthetic cathinones are increasingly popular new psychoactive substances (NPS), with new structures often designed to evade legal restrictions. Besides presenting challenges for drug policy, this also results in a poor understanding of these compounds’ pharmacological effects, particularly concerning their interaction with monoamine transporters such as the serotonin transporter (SERT). Therefore, we optimized a previously developed SERT bioassay based on the TRACT (transporter activity through receptor activation) principle by developing a new human embryonic kidney (HEK) 293T cell line stably expressing SERT (HEK293T-SERT). The use of this cell line was evaluated in the TRACT protocol to determine uptake inhibition and SERT-mediated serotonin (5-HT) release. SERT effects are measured by means of a modified serotonin 2&#xa0;A receptor (5-HT<sub>2A</sub>R), using functional complementation of a split-nanoluciferase. 5-HT<sub>2A</sub>R activation depends on extracellular serotonin levels, which are elevated either through inhibited or reversed 5-HT transport. The TRACT assay format for SERT inhibition with the new HEK293T-SERT cell line outperformed the transient transfection protocol. In addition, the use of the HEK293T-SERT cells in a fluorescence-based functional assay resulted in an identical ranking of the SERT inhibition activities of a set of eleven diversely substituted cathinones in both TRACT and fluorescence assays. These compounds carried key substitutions, including <i>N</i>-alkylation/arylation, ring substitution and α-alkylation. Furthermore, the TRACT assay format was suited to distinguish between SERT blockers and releasers, and allowed functional characterization. Overall, the dual functionality of the optimized TRACT assay provides a versatile tool for characterizing SERT-modulating NPS, with potential applications in therapeutic drug development.</p>

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TRACT assay-based in vitro characterization of substituted cathinones: inhibition and release at the serotonin transporter

  • 期刊论文

  • 发表日期：

    2026年3月31日

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  • Axelle Timmerman,
  • Eline Pottie,
  • Christophe P. Stove

摘要

Synthetic cathinones are increasingly popular new psychoactive substances (NPS), with new structures often designed to evade legal restrictions. Besides presenting challenges for drug policy, this also results in a poor understanding of these compounds’ pharmacological effects, particularly concerning their interaction with monoamine transporters such as the serotonin transporter (SERT). Therefore, we optimized a previously developed SERT bioassay based on the TRACT (transporter activity through receptor activation) principle by developing a new human embryonic kidney (HEK) 293T cell line stably expressing SERT (HEK293T-SERT). The use of this cell line was evaluated in the TRACT protocol to determine uptake inhibition and SERT-mediated serotonin (5-HT) release. SERT effects are measured by means of a modified serotonin 2 A receptor (5-HT2AR), using functional complementation of a split-nanoluciferase. 5-HT2AR activation depends on extracellular serotonin levels, which are elevated either through inhibited or reversed 5-HT transport. The TRACT assay format for SERT inhibition with the new HEK293T-SERT cell line outperformed the transient transfection protocol. In addition, the use of the HEK293T-SERT cells in a fluorescence-based functional assay resulted in an identical ranking of the SERT inhibition activities of a set of eleven diversely substituted cathinones in both TRACT and fluorescence assays. These compounds carried key substitutions, including N-alkylation/arylation, ring substitution and α-alkylation. Furthermore, the TRACT assay format was suited to distinguish between SERT blockers and releasers, and allowed functional characterization. Overall, the dual functionality of the optimized TRACT assay provides a versatile tool for characterizing SERT-modulating NPS, with potential applications in therapeutic drug development.