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 [H-3] 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, [H-3] 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 muM) was approximately 10-fold higher in HeLa/hPept1 cells. Also, Lys-FITC-OCH3 (100 muM) 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 x 10(6) 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.