Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors

Scientific Reports, vol. 11, pp. 12290

Abstract

The human norepinephrine transporter (NET) is an established drug target 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. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET-norepinephrine (NE), dopamine (DA) and epinephrine (EP)-were compared in the characterization of the reference NET inhibitor nisoxetine. 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. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

Authors 8

1. Hubert J. Sijben Aachen Division of Drug Discovery and Safety

   Leiden University

   Affiliation as printed

   Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands

2. Wieke M. van Oostveen Aachen Division of Drug Discovery and Safety

   Leiden University

   Affiliation as printed

   Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands

3. Peter B. R. Hartog Aachen Division of Drug Discovery and Safety

   Leiden University

   Affiliation as printed

   Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands

4. Laura Stucchi

   Axxam (Italy)

   Affiliation as printed

   Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy

5. Andrea Rossignoli

   Axxam (Italy)

   Affiliation as printed

   Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy

6. Lia Scarabottolo

   Axxam (Italy)

   Affiliation as printed

   Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy

7. Adriaan P. IJzerman Aachen Division of Drug Discovery and Safety

   Leiden University

   Affiliation as printed

   Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands

8. Laura H. Heitman corresponding Aachen Division of Drug Discovery and Safety

   Leiden University · Oncode Institute

   Affiliation as printed

   Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands. l.h.heitman@lacdr.leidenuniv.nl

   Oncode Institute, Leiden, The Netherlands. l.h.heitman@lacdr.leidenuniv.nl

   Oncode Institute, Leiden, The Netherlands