Transport of amino acid aryl amides by the intestinal H⁺/peptide cotransport system, PEPT1

Abstract

Transport of amino acid aryl amides by the intestinal H ϩ /peptide symporter (PEPT1) was studied in Caco-2 cells and in Xenopus laevis oocytes expressing human PEPT1. Several amino acid amides were able to inhibit the uptake of [ 14 C]glycylsarcosine in Caco-2 cells. Ala-4-nitroanilide (K i ϭ 0.08 mM), Phe-4-nitroanilide (K i ϭ 0.09 mM) and Ala-4-phenylanilide (Ki ϭ 0.03 mM) were accepted as substrates with equal or higher affinity than natural Ala-Xaa dipeptides. Ala-anilide (K i ϭ 2.9 mM), Ala-7-amido-4-methylcoumarin (K i ϭ 0.2 mM), Ala-4-chloroanilide (K i ϭ 0.3 mM) and Ala-4-methylanilide (K i ϭ 0.3 mM) were also recognized by PEPT1 as substrates. In contrast, alanine, Ala-amide, Phe-amide, Ala-methyl ester, Ala-4-nitrobenzyl ester and Ala-methylamide were not recognized (K i Ͼ 20 mM). In X. laevis oocytes, transport of Ala-4-nitroanilide, Ala-7-amido-4-methylcoumarin, Ala-4-methylanilide and Alaanilide was associated with transfer of positive charge and the currents were saturable with respect to substrate concentration (K 0.5 values : 0.1, 0.2, 0.8 and 3.1 mM, respectively). The currents induced by Ala-4-methylanilide were saturable with respect to the substrate concentration and influenced by the membrane potential. The affinity of the transporter for Ala-4-methylanilide was also found to be influenced by the membrane potential. We conclude that the intestinal H ϩ /peptide cotransport system PEPT1 accepts amino acid aryl amides as substrates.Keywords : intestinal peptide transport; PEPT1; Caco-2 cells; amino acid amide; electrophysiology.The H ϩ /peptide cotransport system PEPT1, expressed in the brush-border membrane of intestinal epithelial cells, accepts small peptides which consist of two or three amino acids as substrates. Free amino acids are excluded by the system [1Ϫ4]. In addition to dipeptides and tripeptides, PEPT1 is also capable of transporting many peptidomimetics such as β-lactam antibiotics, angiotensin-converting enzyme inhibitors and bestatin analogues [5,6]. The structural requirements for substrates to be accepted by peptide transporters are not fully understood. Many, yet not all of the diverse substrates studied so far, share structural features such as a peptide bond with an A-amino group and a C-terminal carboxylic acid group [5Ϫ7]. They all seem to bear, however, sterical resemblance to the backbone of physiologically occurring dipeptides and tripeptides. Data concerning the minimal structural requirements for substrate recognition are a prerequisite for the design of pharmacologically active peptidomimetics that are transported by peptide carriers and can, therefore, be administered orally.The purpose of the present investigation was to study in a systematic approach the basic structural requirements of peptide Correspondence to M. Brandsch, Biozentrum of the Martin-LutherUniversity Halle-Wittenberg, c

An update on renal peptide transporters

“…Large sets of dipeptides, amino acid derivatives, or peptidomimetics have been probed for transport in competition experiments utilizing cells expressing either one of the transporters, by the measurement of substrate-mediated transport currents in Xenopus laevis oocytes expressing PEPT1 or PEPT2, or by competition experiments in the yeast Pichia pastoris heterologously expressing either one of the mammalian transporters (11,12,14,21,22,66,67). One important finding for understanding the "multispecificity" of the peptide transporters was that neither PEPT1 nor PEPT2 requires a peptide bond in a substrate and that the minimal structural requirement in a substrate for binding and transport is a simple carbon chain that separates the oppositely charged NH 2 and COOH head groups by an intramolecular distance of Ͼ 500 Ͻ 630 picometers (21).…”

Defining Minimal Structural Features in Substrates of the H+/Peptide Cotransporter PEPT2 Using Novel Amino Acid and Dipeptide Derivatives

“…The intestinal peptide transporter PEPT1, belonging to the same family as PEPT2, mediates the uptake of di-and tripeptides and derivatives into intestinal epithelial cells in a similar mode. PEPT1 has been studied extensively with respect to substrate specificity, and findings allowed the formulation of preliminary predictive models for substrate recognition (Borner et al, 1998;Brandsch et al, 1998Brandsch et al, , 1999Döring et al, 1998d;Meredith et al, 1998). In previous studies (Döring et al, 1998d), we showed that PEPT1 transports -amino fatty acids electrogenically with affinities that are similar to those of native dipeptides.…”

“…Recent findings demonstrated that amino acid arylamides completely lacking a terminal carboxylic group serve as substrates for PEPT1 (Borner et al, 1998). To probe whether this also applies to PEPT2, we used a set of para-substituted alanine-anilides having the structures provided in Fig.…”

“…By using -amino fatty acids of increasing chain length, the minimal molecular requirements for substrate-PEPT1 interactions were defined as free terminal amino and carboxylic groups separated by at least four methylene groups. Another study demonstrated that amino acid arylamides are recognized by PEPT1 as highaffinity substrates (Borner et al, 1998) and that the replacement of the native peptide bond by a thioxo-peptide bond is also well-tolerated by PEPT1 . These and other data obtained in different expression systems for PEPT1 of different species were used for the first modeling approaches to obtain a template for the interaction within the substrate-binding domain (Bailey et al, 2000).…”

Interaction of Intestinal Nucleoside Transporter hCNT2 with Amino Acid Ester Prodrugs of Floxuridine and 2-Bromo-5,6-dichloro-1-.BETA.-D-ribofuranosylbenzimidazole

“…[7][8][9] Enhanced bioavailabilities of the amino acid prodrugs is attributed to carrier mediated intestinal absorption via intestinal transporters such as a human peptide transporter hPept1 or an amino acid transporter ATB 0,ϩ . 4,[10][11][12] Nucleoside antiviral and anticancer agents are important class of drugs for the fight against viral diseases and cancer. However, they often exhibit poor pharmaceutical and absorption, distribution, metabolism and excretion (ADME) properties.…”