This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.

The maltose transport system of Escherichia coli is a well‐characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein‐dependent transport systems, like the maltose transport system of E.coli, possess a water‐soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane‐bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein‐independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P‐glycoprotein or CFTR, that are implicated in disease.

We have used epitope insertion to analyze the transmembrane topology of the human H+-dipeptide symporter hPEPT1. An epitope tag, EYMPME (EE), was inserted into different locations at amino acids 39, 78, 106, 412, and 708 of hPEPT1 by site-directed mutagenesis. The functional integrity of the tagged protein was tested by measuring its dipeptide transport activity in transfected Cos7 cells. Further, cells expressing hPEPT1 or EE-tagged hPEPT1 derivatives were labeled with an anti-EE-monoclonal antibody (anti-EE-mAb) or an antiserum raised against the carboxyl terminus of hPEPT1 (anti-hPEPT1) and examined by immunofluorescence confocal microscopy. EE106-, 412-, and 708-hPEPT1 transported the dipeptide tracer as well as wild-type hPEPT1. Tags at position 106 and 412 were shown to be extracellular because they were accessible to anti-epitope antibody in nonpermeabilized cells. In contrast, the carboxyl-terminal domain and EE708 were shown to be intracellular since they were only accessible to the antibodies in permeabilized cells. These results are consistent with a 12-transmembrane domain (TMD) topological model of PEPT1. Epitope insertions at regions linking the putative TMD1 and TMD2, and TMD2 and TMD3 (EE39- and EE78-hPEPT1), abolished the dipeptide transport into the cells. In transfected Cos7 cells, these tagged proteins remained largely intracellular rather than at the plasma membrane. These results suggest that the integrity of these regions is essential for transporter trafficking and/or function. Thus, the topology of the amino-terminal portion, including putative TMD1 and -2, remains to be clarified.