Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1

Abstract

Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs), transporter inhibition may affect GPCR signalling due to a change in extracellular concentration of the substrate/ligand, which can be monitored by an impedance-based label-free assay. For this study, a prototypical SLC/GPCR pair was selected, i.e. the equilibrative nucleoside transporter-1 (SLC29A1/ENT1) and an adenosine receptor (AR), for which adenosine is the substrate/ligand. ENT1 inhibition with three reference compounds was monitored sensitively via AR activation on human osteosarcoma cells. Firstly, the inhibitor addition resulted in an increased apparent potency of adenosine. Secondly, all inhibitors concentration-dependently increased the extracellular adenosine concentration, resulting in an indirect quantitative assessment of their potencies. Additionally, AR activation was abolished by AR antagonists, confirming that the monitored impedance was AR-mediated. In summary, we developed a novel assay as an in vitro model system that reliably assessed the potency of SLC29A1 inhibitors via AR signalling. As such, the method may be applied broadly as it has the potential to study a multitude of SLCs via concomitant GPCR signalling.

Peptide-Based Strategies for Targeted Tumor Treatment and Imaging

“…Peptides of synthetic or natural origins are, however, amenable to structural modifications owing to their poor pharmacokinetics. Thus, the target binding sequence motifs have been identified and validated after structure–activity relationship (SAR) profiling using several functional assays and sequence algorithm-based programs, such as BLAST homology search, in silico analysis, and SRMAtlas [ 1 , 2 , 3 , 4 , 5 ]. Given the natural and artificial sources of targeting peptides, it is essential that tumors and/or their associated microenvironment, including vascular cells, extracellular matrix, and immune cells overexpress accessible, specific and functional biomarkers for efficient targeting and imaging.…”

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

“…The TRACT assay principle assumes that the substrate of the transporter (NET) is able to induce a cellular response (e.g., by activation of a cell surface GPCR), where the transporter activity (i.e., uptake of substrate) indirectly affects the magnitude of the substrate-induced response, as has been shown recently 20 . Besides its main substrate, NE, NET is known to transport the catecholamines DA and EP 33,34 as well as other amines and substances such as tyramine, phenylethylamine and MPP+ 6 .…”

“…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 . Recently, our group developed a novel functional 'transport activity through receptor activation' (TRACT) assay based on a label-free impedance-based technology for the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) 20,21 and the dopamine transporter (DAT, SLC6A3) 22 . In this bioassay a transporter that shares its substrate (e.g., adenosine, dopamine) with a G protein-coupled receptor (GPCR) is expressed in live cells together with a cognate GPCR.…”

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

“…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 . Recently, our group developed a novel functional ‘transport activity through receptor activation’ (TRACT) assay based on a label-free impedance-based technology for the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) 20 , 21 and the dopamine transporter (DAT, SLC6A3) 22 . In this bioassay a transporter that shares its substrate (e.g., adenosine, dopamine) with a G protein-coupled receptor (GPCR) is expressed in live cells together with a cognate GPCR.…”