Transport of the phosphonodipeptide alafosfalin by the H⁺/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells

Abstract

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H + /peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [ 14 C]glycylsarcosine with K i values of 0.19 ± 0.01 mM and 0.07 ± 0.01 mM for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K t ) but not the maximal velocity (V max ) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [ 14 C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mM) by 73%. In SKPT cells, uptake of [ 14 C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [ 14 C]Gly-Sar by alafosfalin. Alafosfalin (3 mM) did not affect the apical to basolateral [ 14 C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells TM , alafosfalin increased the shortcircuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H + /peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H + -symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H + /peptide cotransporters.Keywords: alafosfalin; alaphosphin; Caco-2 cells; SKPT cells; Ussing technique.Alafosfalin (alaphosphin, L-alanyl-L-1-aminoethylphosphonic acid) is an antibacterial dipeptide analogue where the carboxyl group at the C-terminal alanine is replaced with a phosphonic [P(O)(OH) 2 ] function. The compound was one of the most promising aminophosphonic acids obtained in an extensive study synthesizing more than 300 di-to penta-peptide alanine mimetics with varying stereometry and different substituents for the C-terminal carboxyl function [1]. It displays good oral availability, substantial antibacterial activity mainly against Gramnegative bacteria and synergism with b-lactam antibiotics [1][2][3][4][5]. In clinical studies alafosfalin was tested for the treatment of gastrointestinal [3] and urinary tract infections [2,4]. Studies demonstrated the competitive effect of food on its enzymatic breakdown in the intestinal lumen [6]. In a recent publication Kafarski & Lejczak [7] reviewed the potential medical importance of aminophosphonic acids and conclude that, due to their negligible mammalian toxicity and the fact that they very efficiently mimic aminocarboxylic acids making them extremely important antimetabolites, aminophosphonic aci...

High-Affinity Interaction of Sartans with H+/Peptide Transporters

“…14 C]Gly-Sar flux (20 M, control) was 1.48 Ϯ 0.1% ⅐ h Ϫ1 ⅐ cm Ϫ2 , which is in agreement with previously published values (Neumann et al, 2004). In the presence of losartan, valsartan, and eprosartan (1 mM), the [ 14 C]Gly-Sar flux was decreased to 0.23 Ϯ 0.01, 0.93 Ϯ 0.05, and 1.31 Ϯ 0.1% ⅐ h Ϫ1 ⅐ cm Ϫ2 , respectively (Fig.…”

“…After incubation for 10 min, the monolayers were quickly washed four times with ice-cold uptake buffer, solubilized, and prepared for liquid scintillation spectrometry. Transepithelial flux of [ 14 C]GlySar across Caco-2 cell monolayers cultured on permeable filters was measured as described previously (Neumann et al, 2004). After washing the inserts at pH 7.5 for 10 min, uptake at 37°C was started by adding 1.5 ml of uptake buffer, pH 6.0, containing [ 14 C]Gly-Sar with or without 1 mM sartan to the donor side.…”

“…We studied the interaction of the four sartans-losartan, irbesartan, valsartan, and eprosartan-with PEPT1 and PEPT2 in competition assays using [ 14 C]Gly-Sar as a reference substrate. The intestinal cell line Caco-2 constitutively expressing PEPT1 (Knütter et al, 2004;Neumann et al, 2004) and the renal cell line SKPT constitutively expressing PEPT2 (Theis et al, 2002;Luckner and Brandsch, 2005) were used. Uptake of [ 14 C]Gly-Sar into Caco-2 cells in the presence of an inwardly directed H ϩ gradient showed concentration-dependent inhibition by losartan, irbesartan, valsartan, and eprosartan (Fig.…”

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

“…S8A). Alafosfalin is a good substrate for POT family transporters (29)(30)(31) and competes with the uptake of the (Ala) 2 peptide by GkPOT ( Fig. 2A).…”

Development of in vitro screening system for food habit related risk analysis

“…Cell and tissue cultures have long been used in medical and pharmaceutical research. Effect of drugs, macromolecules and other substances on cell absorption and transportation properties are already being studied in cell cultures (Artursson & Karlsson, 1991;Uil et al, 1997;Neumann et al, 2004;Weng et al, 2005;Grunwald et al, 2006;Cardinali et al, 2013;Jarmolowska et al, 2013).…”