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.
Highlights
Solute carriers (SLCs) are transmembrane transport proteins that control a cell’s intra- and extracellular communication with its environment by regulating the translocation of small molecules, inorganic ions or other proteins across biological membranes
No label-free assays applicable to non-electrogenic membrane transporters, which represent the majority of Solute Carrier (SLC) are available
ENT1 is the most abundant nucleoside transport (NT) protein in the human body[13]. This SLC plays a crucial role in the provision of nucleosides, as it participates in the salvage pathways of nucleotide synthesis in cells lacking de novo biosynthetic pathways[14]
Summary
Solute carriers (SLCs) are transmembrane transport proteins that control a cell’s intra- and extracellular communication with its environment by regulating the translocation of small molecules, inorganic ions or other proteins across biological membranes Their function as gatekeepers, their ubiquitous presence in the human body, and the large number of SLCs (more than 400) render them potential therapeutic targets[1]. Their in-depth investigation has been limited as not many cellular assays are available to study transporter activity, making drug discovery difficult. We describe the development of a novel cellular assay for the functional assessment of SLC activity by using the label-free impedance-based xCELLigence instrument. For validation purposes we performed radioligand binding studies on SLC29A1 as well
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