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

Abstract

A major challenge in drug development is the optimization 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 recognize a diverse library of di- and tripeptides, making them extremely promiscuous and major contributors to the pharmacokinetic profile of several important drug classes, including beta-lactam antibiotics and antiviral and antineoplastic agents. Of particular interest has been their ability to recognize 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 homolog 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 with physiological peptides. These structures provide a unique insight into how peptide transporters interact with xenobiotic molecules and provide a template for further prodrug development.

Keywords: SLC15; drug transport; membrane transport; proton-coupled transport.

Fig. 1.

Crystal structure of PepT_Sh in complex with valacyclovir. (A) PepT_Sh represented as light blue helices in the plane of the membrane. The binding cavity surface is colored according to the localized electrostatic potential. Yellow sticks represent the bound prodrug, valacyclovir. Waters are shown as red spheres. (B) Residues that interact with valacyclovir are shown as blue sticks. Hydrogen bonds are shown as red dashes and distances are labeled. (Inset Left) Experimental mFo-DFc difference electron density (green mesh) for valacylcovir, contoured at 3 σ. (Inset Right) Final refined 2mFo-DFc electron density (blue mesh), contoured at 1 σ.

Fig. 2.

Functional analysis of PepT_Sh binding site variants. Schematic of valacyclovir (green) interacting with PepT_Sh (black). The contribution of each interacting residue was analyzed using IC₅₀ competition assays using three substrates: AlaAla, AlaAlaAla, and valacyclovir are shown. The results are plotted as a bar graph for each variant and compared against WT (blue bars). Interatomic distances (in angstroms) are shown in red.

Fig. 3.

Crystal structure of PepT_Sh in complex with 5-aminolevulinic acid. (A) Five-aminolevulinic acid (5-ALA, orange sticks) bound to PepT_Sh (green). Hydrogen bond interactions are shown as red dashes with distances indicated. Water molecules are shown as red spheres. Key residues involved in binding substrate are shown as green sticks. (Inset Left) Experimental mFo-DFc difference electron density (green mesh) observed for 5-aminolevulinic acid, contoured at 3 σ. (Inset Right) Final refined 2mFo-DFc electron density map (blue mesh), contoured at 1 σ. (B) Schematic of 5-aminolevulinic acid (purple) interacting with PepT_Sh (black). Nearby residues that are not interacting with the ligand are indicated in gray. IC₅₀ values for the two variants tested are shown as bar charts and compared with WT values. Interatomic distances (in angstroms) are shown in red.

Fig. 4.

Pharmacophore model for valacyclovir binding to POT family transporters. (A) Key interaction sites observed in PepT_Sh are shown for valacyclovir in the context of the full transporter structure. (B) Closeup view of the PepT_Sh binding site accommodating valacyclovir. (C) Proposed pharmacophore model indicating the role of conserved SLC15 family binding site residues in recognizing either peptides (AlaPhe) (PDB: 4D2C) or prodrug (valacyclovir).