Advantages and shortcomings of cell-based electrical impedance measurements as a GPCR drug discovery tool

Doijen, Jordi; Loy, Tom Van; Landuyt, Bart; Luyten, Walter; Schols, Dominique; Schoofs, Liliane

doi:10.1016/j.bios.2019.04.041

Cited by 22 publications

(25 citation statements)

References 132 publications

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“…71,72 GPCRs are the largest superfamily of cell-surface receptors involved in TMD signalling, usually transmitting signals into cells via their response to a range of extracellular stimuli, such as glycoproteins, polypeptides and ions, and hence regulating a wide variety of physiological and developmental functions. 73 The intracellular-signalling cascades activated by GPCRs have been proven to be remarkably complex. 73,74 The binding of a ligand to the GPCR binding site leads to a conformational change in the receptor, in turn promoting the binding of the heterotrimeric G proteins, consisting of G a -GDP and G bgsubunits, within the intracellular moiety of the receptor.…”

Section: Allosteric Binding and Cooperativitymentioning

confidence: 99%

“…73 The intracellular-signalling cascades activated by GPCRs have been proven to be remarkably complex. 73,74 The binding of a ligand to the GPCR binding site leads to a conformational change in the receptor, in turn promoting the binding of the heterotrimeric G proteins, consisting of G a -GDP and G bgsubunits, within the intracellular moiety of the receptor. 74 The exchange of GTP for GDP on the G a -subunit results in the reversible dissociation of the G protein subunits, initiating a downstream signalling via G a -GTP and G bg .…”

Section: Allosteric Binding and Cooperativitymentioning

confidence: 99%

“…74 The exchange of GTP for GDP on the G a -subunit results in the reversible dissociation of the G protein subunits, initiating a downstream signalling via G a -GTP and G bg . 73,74…”

Section: Allosteric Binding and Cooperativitymentioning

confidence: 99%

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G-Protein coupled receptors: structure and function in drug discovery

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Section: Allosteric Binding and Cooperativitymentioning

confidence: 99%

Section: Allosteric Binding and Cooperativitymentioning

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“…In this study, a label-free TRACT assay was developed and validated for the human NET using an impedance-based biosensor, xCELLigence 23 – 25 . To develop the assay we used a modified human embryonic kidney (HEK) 293 cell line with doxycycline-inducible overexpression of NET and endogenous expression of adrenergic receptors (JumpIn-NET).…”

Section: Introductionmentioning

confidence: 99%

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

Sijben

Oostveen

Hartog

et al. 2021

Sci Rep

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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.

show abstract

“…In this study, a label-free TRACT assay was developed and validated for the human NET using an impedance-based biosensor, xCELLigence [23][24][25] . To develop the assay we used a modified human embryonic kidney (HEK)-293 cell line with doxycycline-inducible overexpression of NET and endogenous expression of adrenergic receptors.…”

Section: Introductionmentioning

confidence: 99%

Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors

Sijben

Oostveen

Hartog

et al. 2021

Preprint

View full text Add to dashboard Cite

The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological 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 inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in 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.

show abstract

Advantages and shortcomings of cell-based electrical impedance measurements as a GPCR drug discovery tool

Cited by 22 publications

References 132 publications

G-Protein coupled receptors: structure and function in drug discovery

G-Protein coupled receptors: structure and function in drug discovery

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

Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors

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