A novel inhibitor of the mammalian peptide transporter PEPT1

This study was initiated to develop inhibitors of the intestinal H+/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO2)]-P ro is an effective competitive inhibitor of mammalian PEPT1 with an apparen t binding affinity of 5-10 muM. Characterization of the interaction of Lys[ Z(NO2)]-Pro with the substrate binding domain of PEPT1 has been performed i n (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transg enic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptid es, HPLC analysis of Lys[Z(NO2)]-Pro in cells, and electrophysiological tec hniques, we unequivocally show that Lys[Z(NO2)]-Pro binds with high affinit y to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated b y the absence of Lys[Z(NO2)]-Pro in Caco-2 cell extracts as determined by H PLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO2)]-Pro can bind to P EPT1 from the extracellular as well. as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced c urrents under voltage clamp conditions. Our findings serve as a starting po int for the identification of the substrate binding domain in the PEPT1 pro tein as well as for studies on the physiological and pharmacological role o f PEPT1.