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Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors.
Sijben, Hubert J; van Oostveen, Wieke M; Hartog, Peter B R; Stucchi, Laura; Rossignoli, Andrea; Maresca, Giovanna; Scarabottolo, Lia; IJzerman, Adriaan P; Heitman, Laura H.
Afiliação
  • Sijben HJ; Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.
  • van Oostveen WM; Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.
  • Hartog PBR; Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.
  • Stucchi L; Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy.
  • Rossignoli A; Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy.
  • Maresca G; Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy.
  • Scarabottolo L; Axxam S.p.A, Openzone Science Park, Bresso, Milan, Italy.
  • IJzerman AP; Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.
  • Heitman LH; Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands. l.h.heitman@lacdr.leidenuniv.nl.
Sci Rep ; 11(1): 12290, 2021 06 10.
Article em En | MEDLINE | ID: mdl-34112854
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fluoxetina / Proteínas da Membrana Plasmática de Transporte de Norepinefrina / Ensaios de Triagem em Larga Escala Tipo de estudo: Diagnostic_studies / Prognostic_studies / Screening_studies Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fluoxetina / Proteínas da Membrana Plasmática de Transporte de Norepinefrina / Ensaios de Triagem em Larga Escala Tipo de estudo: Diagnostic_studies / Prognostic_studies / Screening_studies Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2021 Tipo de documento: Article