Development of norepinephrine transporter reuptake inhibition assays using SK-N-BE(2)C cells

Decker, Ann M.; Blough, Bruce E.

doi:10.1016/j.heliyon.2018.e00633

Cited by 3 publications

(4 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although information on uptake kinetics could provide a more substantiated explanation to this, a drawback of the TRACT assay is that it cannot be used to directly measure the substrate uptake kinetics (e.g., K m , V max ). Nevertheless, the inhibitory potency values of all tested inhibitors were in line with previously reported values from both fluorescent substrate uptake assays 17 , 18 , 46 , 47 and radioligand uptake assays 16 – 18 , 48 – 51 indicating that the TRACT assay can be reliably used for NET inhibitor characterization.…”

Section: Discussionsupporting

confidence: 88%

“…Conventionally, in vitro methods to functionally characterize NET inhibitors are based on inhibition of uptake of a radiolabeled substrate (e.g., [ 3 H]norepinephrine 15 , 16 ) or a fluorescent substrate 17 , 18 . While these methods generally provide reliable IC 50 values for NET inhibitors, the use of labeled substrates has practical downsides such as high costs, waste management, safety precautions and availability of suitable (fluorescent) substrates, limiting the broad implementation of these principles for drug screening 19 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Sijben

Oostveen

Hartog

et al. 2021

Sci Rep

View full text Add to dashboard Cite

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

Section: Discussionsupporting

confidence: 88%

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

View full text Add to dashboard Cite

show abstract

“…Nevertheless, the inhibitory potency values of all tested inhibitors were in line with previously reported values from both fluorescent substrate uptake assays 17,18,42,43 and radioligand uptake assays [16][17][18][44][45][46][47] indicating that the TRACT assay can be reliably used for NET inhibitor characterization.…”

Section: Discussionsupporting

confidence: 87%

“…Conventionally, in vitro methods to functionally characterize NET inhibitors are based on inhibition of uptake of a radiolabeled substrate (e.g., [ 3 H]norepinephrine 15,16 ) or a fluorescent substrate 17,18 . While these methods generally provide reliable IC 50 values for NET inhibitors, the use of labeled substrates has practical downsides such as high costs, waste management, safety precautions and availability of suitable (fluorescent) substrates, limiting the broad implementation of these principles for drug screening 19 .…”

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

Rationalizing the Binding Modes of PET Radiotracers Targeting the Norepinephrine Transporter

et al. 2023

View full text Add to dashboard Cite

Purpose: A new PET radiotracer 18F-AF78 showing great potential for clinical application has been reported recently. It belongs to a new generation of phenethylguanidine-based norepinephrine transporter (NET)-targeting radiotracers. Although many efforts have been made to develop NET inhibitors as antidepressants, systemic investigations of the structure–activity relationships (SARs) of NET-targeting radiotracers have rarely been performed. Methods: Without changing the phenethylguanidine pharmacophore and 3-fluoropropyl moiety that is crucial for easy labeling, six new analogs of 18F-AF78 with different meta-substituents on the benzene-ring were synthesized and evaluated in a competitive cellular uptake assay and in in vivo animal experiments in rats. Computational modeling of these tracers was established to quantitatively rationalize the interaction between the radiotracers and NET. Results: Using non-radiolabeled reference compounds, a competitive cellular uptake assay showed a decrease in NET-transporting affinity from meta-fluorine to iodine (0.42 and 6.51 µM, respectively), with meta-OH being the least active (22.67 µM). Furthermore, in vivo animal studies with radioisotopes showed that heart-to-blood ratios agreed with the cellular experiments, with AF78(F) exhibiting the highest cardiac uptake. This result correlates positively with the electronegativity rather than the atomic radius of the meta-substituent. Computational modeling studies revealed a crucial influence of halogen substituents on the radiotracer–NET interaction, whereby a T-shaped π–π stacking interaction between the benzene-ring of the tracer and the amino acid residues surrounding the NET binding site made major contributions to the different affinities, in accordance with the pharmacological data. Conclusion: The SARs were characterized by in vitro and in vivo evaluation, and computational modeling quantitatively rationalized the interaction between radiotracers and the NET binding site. These findings pave the way for further evaluation in different species and underline the potential of AF78(F) for clinical application, e.g., cardiac innervation imaging or molecular imaging of neuroendocrine tumors.

show abstract

Development of norepinephrine transporter reuptake inhibition assays using SK-N-BE(2)C cells

Cited by 3 publications

References 16 publications

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

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

Rationalizing the Binding Modes of PET Radiotracers Targeting the Norepinephrine Transporter

Contact Info

Product

Resources

About