Members of the solute carrier (SLC) transporter protein family are increasingly recognized as viable therapeutic targets. Although some members are successful drug targets, the vast majority of SLCs have not been exploited therapeutically. The human norepinephrine transporter (NET) is one of these established SLC drug targets for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. To aid in SLC drug development, we have recently developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay.<br/>In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK293-JumpIn cell line. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z’ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. Next, a predictive model for NET was created using publicly available bioactivity data on NET and related proteins. Virtual screening was performed with this model to find untested synthesizable compounds. This resulted in a final set of 46 representative compounds, including several novel and chemically dissimilar NET inhibitors. From this set 32 compounds were synthesized and screened in the TRACT assay at 10 µM. The inhibitory potencies of the top five hits were assessed in the TRACT assay and proved to be modestly active with a pIC50 value of 7.6 ± 0.1 for the most potent compound.<br/>In conclusion, these findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors to provide novel chemical matter for drug discovery. Importantly, this study provides a basis for investigation of other solute carrier transporters with label-free biosensors.

Novel ways to investigate SLC transporter function for drug discovery – a case for the Norepinephrine transporter (NET/SLC6A2)

ACS Spring 2022

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