A Fluorescent hPept1 Transporter Substrate for Uptake Screening

Purpose. To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH₃, Lys-FITC-OH, and Lys-FITC-OCH₃, and to characterize their hPept1 transporter-mediated uptake. Methods. FITC analogues of amino acids were synthesized using established synthetic procedures, and the extent of their [³H]Gly-Sar uptake inhibition in HeLa/hPept1 cells was determined. The uptake of Lys-FITC-OCH₃ was evaluated in HeLa, HeLa/hPept1, and Caco-2 cells in the presence and absence of Gly-Sar using a fluorescence microscopy-based assay. The uptake and transport of the Lys-FITC analogues were also determined in Caco-2 cells using HPLC assays. Results. In HeLa/hPept1 cells, [³H]Gly-Sar uptake was significantly inhibited by Lys-FITC-OCH₃ (74%) but not by FITC-Val-OCH₃ (22%). The uptake of Lys-FITC-OCH₃ (100 μM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, LyS-FITC-OCH₃ (100 μM) uptake in HeLa/hPept1 and Caco-2 cells was reduced by 77% and 80%, respectively, in the presence of 1 mM Gly-Sar. Dipeptides and cephalexin significantly reduced Lys-FITC-OCH₃ uptake in Caco-2 cells. The apical permeability of Lys-FITC-OCH₃ (1.5 × 10⁶ cm/s) in Caco-2 cells was significantly lowered in the presence of Gly-Sar. Fluorescence micrographs revealed that this analogue was localized in the cytoplasm and in the nucleus. Conclusions. The combined results indicate that Lys-FITC-OCH₃ is recognized and transported by hPept1 in HeLa/hPept1 and by peptide transporters in Caco-2 cells. The results also suggest that Lys-FITC-OCH₃ might be a useful fluorescent substrate for rapid assessment of peptide transporter activity in cells of interest.