Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships

Abstract

Chemical modification of proteins is a vintage strategy that is still fashionable due to the information that can be obtained from this approach. An interesting application of chemical modification is linked with membrane transporters. These proteins have peculiar features such as the presence of hydrophobic and hydrophilic domains, which show different degree of accessibility to chemicals. The presence of reactive residues in the membrane transporters is at the basis of the chemical targeting strategy devoted to investigating structure/function relationships; in particular, information on the substrate binding site, regulatory domains, dimerization domains, and the interface between hydrophilic loops and transmembrane domains has been obtained over the years by chemical targeting. Given the difficulty in handling membrane transporters, their study experienced a great delay, particularly concerning structural information. Chemical targeting has been applied with reasonable success to some membrane transporters belonging to the families SLC1, SLC6, SLC7, and SLC22. Furthermore, some data on the potential application of chemical targeting in pharmacology are also discussed.

Figure 1

Chemical targeting of membrane transporters. The sketch depicts a typical asymmetric transporter that, upon conformational changes, exposes the substrate site alternatively to the external or internal side of the membrane. The reagent, depicted as a starred “R”, can have different size as well as degree of hydrophilicity/hydrophobicity. The target, indicated by T, can be any lateral group of an amino acid such as a thiol residue (SH) of cysteine as well as a NH₂ of lysine, etc. The T group exhibits four different typical exposures: (1) T facing the hydrophilic protein exterior (blue) that can be targeted by large hydrophilic reagents; (2) T facing the internal hydrophilic transport path (purple) that can be targeted by small hydrophilic reagents; (3) T facing the intracellular hydrophilic surface (green) that can be targeted by hydrophobic membrane permeant reagents; and (4) T facing the hydrophobic protein moiety in contact with the membrane (red) that can be targeted by hydrophobic small reagents. Effects observed upon targeting can then be ascribed to the targeting of a specific protein moiety based on the type of reagent.

Figure 2

Chemical targeting coupled to site-directed mutagenesis. The sketch depicts a typical asymmetric transporter inserted in a cell membrane. Targetable residues (T) or their mutants (M) are exposed to the reagent (R). (A) In the wild-type transporter, after the interaction with the reagent, a measurable effect called “ON” is observed. (B) In the mutant protein, in which one of the targetable residues (T) is substituted by a nontargetable residue (M), the effect observed in (A) disappears (“OFF”), if the mutated residue is actually the target of the reagent.

Figure 3

Prediction of the hOCTN1 structure. Topology 2D model obtained using the Kyte–Doolittle algorithm for hydropathy analysis of the hOCTN1 protein. Light blue barrels indicate 12 hydrophobic transmembrane spanning domains. Hydrophilic loops connecting transmembrane domains are depicted in green. The seven cysteine residues are numbered and depicted as yellow ovals. The N- and C-termini of the protein face toward the intracellular side. From the 2D model, the 3D homology model of the hOCTN1 protein is built. The transmembrane domains are represented as a light blue ribbon, while the loops connecting the domains are in green. The 3D model has been built using a Phyre2 Server on the structure of the eukaryotic phosphate transporter from P. indica (4J05) as a template. The side chains of the 7 Cys residues are highlighted by numbered balls and sticks.

Figure 4

Dithiazole molecules. (A) The scaffold molecule responsible for interaction with rat ASCT2 and a list of substituents used for the most potent inhibitors. (B) Structures of the two most potent inhibitors able to block human LAT1 with highlighted substituents (in red) of the scaffold dithiazole.