Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system

J Pharmacol Toxicol Methods. 2018 Jul-Aug:92:67-76. doi: 10.1016/j.vascn.2018.03.004. Epub 2018 Mar 23.


Introduction: Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purification tasks even more challenging compared to soluble proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concentrated protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates.

Method: The originality of our approach is based on the comparative analysis of solubilized total membrane preparations with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomolecules in solution to migrate along a temperature gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labelled SLC molecules in the presence of different ligand concentrations.

Results: We herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purification step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 μM.

Discussion: This novel LBA approach is independent of protein purification. It is suitable for drug discovery as it is upscalable to high throughput compound screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward "deorphanization" of SLCs, revealing their transport substrates.

Keywords: Compound screening; Drug discovery; Ligand-binding assay (LBA); Membrane transport; Microscale thermophoresis (MST); Solute carrier family SLC.

Publication types

  • Validation Study

MeSH terms

  • Acyclovir / analogs & derivatives*
  • Acyclovir / metabolism
  • Biological Transport
  • Dipeptides / metabolism*
  • Drug Discovery / methods
  • High-Throughput Screening Assays / methods
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Ligands
  • Peptide Transporter 1 / metabolism*
  • Protein Binding
  • Saccharomyces cerevisiae / metabolism
  • Solute Carrier Proteins / metabolism*
  • Temperature
  • Valacyclovir
  • Valine / analogs & derivatives*
  • Valine / metabolism


  • Dipeptides
  • Ligands
  • Peptide Transporter 1
  • Solute Carrier Proteins
  • glycylsarcosine
  • Valine
  • Valacyclovir
  • Acyclovir