A Quantitative Structure–Activity Relationship for Translocation of Tripeptides via the Human Proton-Coupled Peptide Transporter, hPEPT1 (SLC15A1)

Abstract

Abstract. The human intestinal proton-coupled peptide transporter, hPEPT1 (SLC15A1), has been identified as an absorptive transporter for both drug substances and prodrugs. An understanding of the prerequisites for transport has so far been obtained from models based on competition experiments. These models have limited value for predicting substrate translocation via hPEPT1. The aim of the present study was to investigate the requirements for translocation via hPEPT1. A set of 55 tripeptides was selected from a principal component analysis based on VolSurf descriptors using a statistical design. The majority of theses tripeptides have not previously been investigated. Translocation of the tripeptides via hPEPT1 was determined in a MDCK/hPEPT1 cell-based translocation assay measuring substrateinduced changes in fluorescence of a membrane potential-sensitive probe. Affinities for hPEPT1 of relevant tripeptides were determined by competition studies with [14 C]Gly-Sar in MDCK/hPEPT1 cells. Forty tripeptides were found to be substrates for hPEPT1, having K m app values in the range 0.4-28 mM. Eight tripeptides were not able to cause a substrate-induced change in fluorescence in the translocation assay and seven tripeptides interacted with the probe itself. The conformationally restricted tripeptide Met-Pro-Pro was identified as a novel high-affinity inhibitor of hPEPT1. We also discovered the first tripeptide (Asp-Ile-Arg) that was neither a substrate nor an inhibitor of hPEPT1. To rationalise the requirements for transport, a quantitative structure-activity relationship model correlating K m app values with VolSurf descriptors was constructed. This is, to our knowledge, the first predictive model for the translocation of tripeptides via hPEPT1.

Ibuprofen is a non‐competitive inhibitor of the peptide transporter hPEPT1 (SLC15A1): possible interactions between hPEPT1 substrates and ibuprofen

“…The recently characterized and validated MDCK/hPEPT1 FLIPR ® membrane potential assay (Molecular Devices, Berkshire, UK) (Faria et al ., 2004; Frolund et al ., 2010; Omkvist et al ., 2010) was used to measure substrate transport via hPEPT1. Gly‐Sar and ibuprofen were applied to the MDCK/hPEPT1 FLIPR ® membrane potential assay in absence or presence of the recently identified high affinity hPEPT1 inhibitor, Met‐Pro‐Pro (Omkvist et al ., 2010). Growth media were aspirated and replaced with 50 µl of FLIPR ® membrane potential assay kit dye stock solution reconstituted in HBSS buffer pH 6.0.…”

“…Translocation via hPEPT1 is electrogenic and changes in membrane potential may thus be used as a surrogate marker for substrate transport. The recently characterized and validated MDCK/hPEPT1 FLIPR ® membrane potential assay (Molecular Devices, Berkshire, UK) Frolund et al, 2010;Omkvist et al, 2010) was used to measure substrate transport via hPEPT1. Gly-Sar and ibuprofen were applied to the MDCK/hPEPT1 FLIPR ® membrane potential assay in absence or presence of the recently identified high affinity hPEPT1 inhibitor, Met-Pro-Pro (Omkvist et al, 2010).…”

“…The area under curve (AUC) of the normalized fluorescence signal was subtracted from the AUC caused by addition of a sample with HBSS, pH 6.0. The AUC values were then expressed as a percentage of the AUC caused by the addition of 20 mM Gly‐Sar (later referred to as response), as described previously (Omkvist et al ., 2010).…”

Quantitative structure‐property relationship modeling of small organic molecules for solar cells applications

“…Alternatively, chemometric and cheminformatic tools commonly used in drug discovery can be used to elucidate quantitative structure‐property relationships (QSPR), as well as to discredit rules only derived from physical principles …”

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

“…The study found that 40 out of 55 tripeptides were translocated, 8 interacted with the assay used, and one (Met-Pro-Pro) was an inhibitor while one (Asp-Ile-Arg) was not recognized by hPEPT1. 31 A series of benzyl esters of the ketomethylene dipeptidomimetics Phe-ψ[COCH 2 ]Asp, Val-ψ[COCH 2 ]-Asp and Asp-ψ[COCH 2 ]Gly were also shown to display affinity for hPEPT1 and were transported through Caco-2 cell monolayers. 14 It could appear that most ligands are also substrates for the transporter, however, none of the antimicrobial di- and tripeptides tested in a recent study were substrates for hPEPT1 despite having moderate affinity towards the transporter for some of the peptides.…”