Abstract
The peptide transporter PEPT-1 (SLC15A1) plays a major role in nutritional supply with amino acids by mediating the intestinal influx of dipeptides and tripeptides generated during food digestion. Its role in the uptake of small bioactive peptides and various therapeutics makes it an important target for the investigation of the systemic absorption of small peptide-like active compounds and prodrug strategies of poorly absorbed therapeutics. The dipeptide glycyl-sarcosine (Gly-Sar), which comprises an N-methylated peptide bond that increases stability against enzymatic degradation, is widely utilized for studying PEPT-1-mediated transport. To support experiments on PEPT-1 inhibitor screening to identify potential substrates, we developed a highly sensitive Gly-Sar quantification assay for Caco-2 cell lysates with a dynamic range of 0.1 to 1000 ng/mL (lower limit of quantification 0.68 nM) in 50 µL of cell lysate. The assay was validated following the applicable recommendations for bioanalytic method validation of the FDA and EMA. Sample preparation and quantification were established in 96-well cell culture plates that were also used for the cellular uptake studies, resulting in a rapid and robust screening assay for PEPT-1 inhibitors. This sample preparation principle, combined with the high sensitivity of the UPLC-MS/MS quantification, is suitable for screening assays for PEPT-1 inhibitors and substrates in high-throughput formats and holds the potential for automation. Applicability was demonstrated by IC50 determinations of the known PEPT-1 inhibitor losartan, the known substrates glycyl-proline (Gly-Pro), and valaciclovir, the prodrug of aciclovir, which itself is no substrate of PEPT-1 and consequently showed no inhibition in our assay.
Highlights
Introduction iationsThe human peptide transporter PEPT-1 (SLC15A1) is predominantly expressed at the apical surface of enterocytes at the brush border membrane of the small intestine and mediates the uptake of dietary dipeptides and tripeptides [1,2,3]
Aprotinin and pepstatin A were purchased from AppliChem (Darmstadt, Germany), leupeptin, aciclovir, and valaciclovir hydrochloride were obtained from Biomol (Hamburg, Germany), whereas pefabloc was received from Carl Roth (Karlsruhe, Germany)
During optimization of collision-induced dissociation (CID), we investigated the four most intense mass transitions (m/z): 147.0 → 90.0 (y1 ), 147.0 → 105.9 (z1 ), 147.0
Summary
The human peptide transporter PEPT-1 (SLC15A1) is predominantly expressed at the apical surface of enterocytes at the brush border membrane of the small intestine and mediates the uptake of dietary dipeptides and tripeptides [1,2,3]. As a high-capacity, low-affinity transporter, PEPT-1 has a high substrate promiscuity and accepts almost all physiological dipeptides and tripeptides as substrates with affinity constants mostly between 0.1 and 0.5 mM [6,7]. PEPT-1 consists of 700–730 amino acids arranged in 12 transmembrane α-helical domains that form a V-shaped transporter [6]. The transporter alternates in a scissor-like motion between an inward-open and outward-open confirmation to enable access to the central substrate binding site from different sides of the Licensee MDPI, Basel, Switzerland.
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