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A fluorescent hPept1 transporter substrate for uptake screening.
Christopher P Landowski1, Hyo-kyung Han, Kyung-Dall Lee
1Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.
Pharmaceutical Research
|December 10, 2003
Summary
Fluorescent analogues of peptide transporter substrates were synthesized and tested. Lys-FITC-OCH3 effectively assesses peptide transporter activity in cells, showing significant uptake and transport by hPept1 and Caco-2 cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Peptide transporters, such as human peptide transporter 1 (hPept1), play a crucial role in the absorption of peptides and peptide-like drugs.
- Developing fluorescent substrates is essential for real-time monitoring of transporter activity and drug uptake.
- Existing fluorescent probes may not accurately reflect the transport mechanisms of specific peptide substrates.
Purpose of the Study:
- To synthesize novel fluorescent analogues of hPept1 substrates, specifically FITC-Val-OCH3, Lys-FITC-OH, and Lys-FITC-OCH3.
- To characterize the uptake of these synthesized analogues mediated by the hPept1 transporter.
- To evaluate the utility of these fluorescent analogues as tools for assessing peptide transporter function.
Main Methods:
- Synthesis of FITC-conjugated amino acid analogues using established chemical procedures.
- Inhibition assays using [3H]Gly-Sar uptake in HeLa/hPept1 cells to assess substrate specificity.
- Fluorescence microscopy and HPLC assays to quantify the uptake and transport of Lys-FITC-OCH3 in various cell lines (HeLa, HeLa/hPept1, Caco-2).
- Assessment of Lys-FITC-OCH3 localization within cells using fluorescence microscopy.
Main Results:
- Lys-FITC-OCH3 significantly inhibited [3H]Gly-Sar uptake in HeLa/hPept1 cells, indicating recognition by the transporter.
- Uptake of Lys-FITC-OCH3 was approximately 10-fold higher in HeLa/hPept1 cells compared to control cells.
- Uptake and apical permeability of Lys-FITC-OCH3 in HeLa/hPept1 and Caco-2 cells were significantly reduced by Gly-Sar, dipeptides, and cephalexin, confirming transporter-mediated transport.
- Fluorescence microscopy revealed cytoplasmic and nuclear localization of Lys-FITC-OCH3.
Conclusions:
- Lys-FITC-OCH3 is recognized and effectively transported by the hPept1 transporter in engineered cells and by endogenous peptide transporters in Caco-2 cells.
- The synthesized fluorescent analogue, Lys-FITC-OCH3, demonstrates potential as a valuable tool for the rapid assessment of peptide transporter activity.
- This fluorescent substrate could facilitate research in drug delivery and understanding peptide transport mechanisms in various cellular models.