phenylalanylalanine and glycylsarcosine

L-Phenylalanyl-Ψ[CS-N]-l-alanine (Phe-Ψ-Ala), a thiourea dipeptide, was evaluated as a probe for peptide transporter 1 (PEPT1). Uptake of Phe-Ψ-Ala in PEPT1-overexpressing HeLa cells was significantly higher than that in vector-transfected HeLa cells and the Km value was 275 ± 32 µM. The uptake was pH-dependent, being highest at pH 6.0, and was significantly decreased in the presence of PEPT1 inhibitors [glycylsarcosine (Gly-Sar), cephalexin, valaciclovir, glycylglycine, and glycylproline]. In metabolism assay using rat intestinal mucosa, rat hepatic microsomes, and human hepatocytes, the amount of Phe-Ψ-Ala was unchanged, whereas phenylalanylalanine was extensively decomposed. The clearance, distribution volume, and half-life of intravenously administered Phe-Ψ-Ala in rats were 0.151 ± 0.008 L/h/kg, 0.235 ± 0.012 L/kg, and 1.14 ± 0.07 h, respectively. The maximum plasma concentration of orally administered Phe-Ψ-Ala (2.31 ± 0.60 µg/mL) in the presence of Gly-Sar was significantly decreased compared with that in the absence of glycylsarcosine (3.74 ± 0.44 µg/mL), suggesting that the intestinal absorption of Phe-Ψ-Ala is mediated by intestinal PEPT1. In conclusion, our results indicate that Phe-Ψ-Ala is a high-affinity, metabolically stable, non-radioactive probe for PEPT1, and it should prove useful in studies of PEPT1, e.g., for predicting drug-drug interactions mediated by PEPT1 in vitro and in vivo.

Topics: Administration, Oral; Animals; Biological Transport; Dipeptides; Half-Life; HeLa Cells; Hepatocytes; Humans; Hydrogen-Ion Concentration; Injections, Intravenous; Intestinal Mucosa; Male; Membrane Transport Modulators; Microsomes, Liver; Peptide Transporter 1; Rats, Wistar; Symporters; Transfection

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.

Topics: Animals; Binding Sites; Biological Transport; Cell Line; Dipeptides; Dogs; Electricity; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Peptide Transporter 1; Proline; Protein Binding; Protein Conformation; Structure-Activity Relationship; Symporters

This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO(2))]-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM. Characterization of the interaction of Lys[Z(NO(2))]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys[Z(NO(2))]-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys[Z(NO(2))]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated by the absence of Lys[Z(NO(2))]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO(2))]-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.

Topics: Animals; Binding, Competitive; Biological Transport, Active; Caco-2 Cells; Carrier Proteins; Dipeptides; Humans; Kinetics; Nitrobenzenes; Oocytes; Patch-Clamp Techniques; Peptide Transporter 1; Pichia; Symporters; Xenopus laevis