Conjugation of dipeptide to fluorescent dyes enhances its affinity for a dipeptide transporter (PEPT1) in human intestinal Caco-2 cells.
Dipeptide transporters in small intestine have a very wide substrate specificity, so that the transporter sometimes serves as a carrier for peptide-like compounds. We have synthesized dipeptide analogues conjugated at an epsilon-amino group of Lys in Val-Lys or Lys-Sar with fluorescent compounds such as fluorescein isothiocyanate and coumarin-3-carboxylic acid. Uptakes of these peptide analogues were examined by measuring intracellular accumulations into monolayers of the human intestinal epithelial cell line Caco-2 expressing the dipeptide transporter PEPT1. Kinetic analysis and effects of addition either of uncoupler (protonophore) or by Gly-Sar, one of the good substrates of PEPT1, revealed that fluorescent dipeptides were taken up by passive diffusion. In contrast, these analogues remarkably inhibited the Gly-Sar uptake by Caco-2 cells. Among the fluorescent analogues synthesized in this paper, Val-Lys(Flu) was the most potent competitive inhibitor against the Gly-Sar uptake with an inhibition constant of 5 microM. This value is the smallest among those ever reported: Val-Lys(Flu) has the highest affinity for PEPT1 among chemicals ever reported. The importance of the hydrophobic part of the substrate was pointed out.
Abe
H
H
Laboratory of Biophysical Chemistry and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812 Japan.
Satoh
M
M
Miyauchi
S
S
Shuto
S
S
Matsuda
A
A
Kamo
N
N
D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D000074011:Peptide Transporter 1; D027981:Symporters
D002352:Carrier Proteins; D004151:Dipeptides; D005456:Fluorescent Dyes; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C074992:valyllysine; C004194:glycylsarcosine; D016650:Fluorescein-5-isothiocyanate