Design, synthesis, and evaluation of tripeptidic promoieties targeting the intestinal peptide transporter hPEPT1

The human intestinal proton coupled di/tri-peptide transporter hPEPT1 promotes the oral bioavailability of several drug compounds. The strategy behind the present work is that by linking a suitable di- or tripeptidic promoiety to a drug substance, by a hydrolysable ester bond, it may give rise to a prodrug that targets hPEPT1. 29 tripeptides were designed based on known structural requirements for substrates binding hPEPT1. Serine, homoserine, or threonine was incorporated in the tripeptide as hydroxy group donors in order for them to be linked to carboxylic drug substances. Optimisation of the promoiety included a study of 14 unnatural tripeptides whose diversity was expressed by VolSurf descriptors. A total of 29 tripeptides was synthesised by solid phase peptide synthesis and a standard Fmoc protocol. The affinity of the tripeptides to hPEPT1 was determined by measuring the inhibition of [(14)C]Gly-Sar in mature Caco-2 cell monolayers which resulted in K(i) values ranging from 0.22 to 25 mM or above. Translocation through the intestinal membrane, mediated by hPEPT1, was measured by recording the membrane potential relative to that induced by the known substrate Gly-Sar. The change in membrane potential is caused by influx of protons due to the co-transport of substrates and protons by hPEPT1 and is, as such, an indication of translocation. A K(i) value of 0.30 mM combined with efficient translocation indicated that H-Phe-Ser-Ala-OH is a suitable lead promoiety for targeted hPEPT1 prodrug design.