Structural basis for prodrug recognition by the SLC15 family of proton coupled peptide transporters

Abstract

A major challenge in drug development is the optimisation of intestinal absorption and cellular uptake. A successful strategy has been to develop prodrug molecules, which hijack solute carrier (SLC) transporters for active transport into the body. The proton coupled oligopeptide transporters, PepT1 and PepT2, have been successfully targeted using this approach. Peptide transporters display a remarkable capacity to recognise a diverse library of di‐ and tri-peptides, making them extremely promiscuous and major contributors to the pharmacokinetic profile of several important drug classes, including beta-lactam antibiotics, anti-viral and antineoplastic agents. Of particular interest has been their ability to recognise amino acid and peptide-based prodrug molecules, thereby providing a rational approach to improving drug transport into the body. However, the structural basis for prodrug recognition has remained elusive. Here we present crystal structures of a prokaryotic homologue of the mammalian transporters in complex with the antiviral prodrug valacyclovir and the peptide based photodynamic therapy agent, 5-aminolevulinic acid. The valacyclovir structure reveals that prodrug recognition is mediated through both the amino acid scaffold and the ester bond, which is commonly used to link drug molecules to the carrier’s physiological ligand, whereas 5-aminolevulinic acid makes far fewer interactions compared to physiological peptides. These structures provide a unique insight into how peptide transporters interact with xenobiotic molecules and provide a template for further prodrug development.

The conformational distribution of a major facilitator superfamily peptide transporter is modulated by the membrane composition

“…POTs have been extensively studied by x-ray crystallography and biochemical transport assays over the past years 27,36,39,59,70,73,83,84 . Numerous structures of various bacterial homologues in the absence and presence of substrates, drugs, and prodrugs are available, highlighting crucial residues for substrate binding and proton coupling [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] . Unfortunately, only inward-open and partially inward-open states of POTs have so far been described at atomic resolution and the influence of the membrane environment on structure and transport has been neglected 41,42,47,52,53,55,58 .…”

“…Their remarkable substrate promiscuity 38,39 together with their highly abundant MFS fold 40 makes them ideal models to understand the interplay between conformational changes and transport. Numerous bacterial POT structures have been determined to date [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] . We focus on the E. coli peptide transporter DtpA 59,60 to explore the effect of different environments such as detergents and Saposin nanoparticles 61,62 (SapNPs) with different lipid compositions on the conformation of the protein using single molecule FRET (smFRET) [63][64][65][66][67][68] .…”

Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

“…Hence, PepT Sh represents an ideal model for understanding the proton coupling mechanism across the bacterial and human homologues with the POT family. Our previous work captured the IF state of PepT Sh bound with S-Cys-Gly-3M3SH as well as several drugs (Minhas et al, 2018;Minhas & Newstead, 2019), although the OF and OC states were not captured. It therefore remains challenging to complete the functional cycle of proton-coupled ligand transport starting from static IF structures.…”

“…Uptake of the S-Cys-Gly-3M3SH ligand into S.hominis results in its biotransformation into an odorous volatile responsible for human body odor (Minhas et al, 2018). PepT Sh shares high sequence similarity with PepT1 and PepT2 and can also transport prodrugs such as valacyclovir and 5-aminolevulinic acid (Minhas et al, 2018;Minhas & Newstead, 2019, 2020. Hence, PepT Sh represents an ideal model for understanding the proton coupling mechanism across the bacterial and human homologues with the POT family.…”