Structural Characterization of Substrate Transport Selectivity of the SLC13 Family of Na+/Dicarboxylate Cotransporters

Abstract

Members of the sodium dependent SLC13 transporter family mediate the transport of di and tricarboxylates of the citric acid cycle into cells. SLC13 transporters such as NaCT (SLC13A5) have been implicated in lifespan extension and resistance to high fat diets and are therefore emerging drug targets for aging and metabolic disorders. Here, we combine various computational and experimental methods to improve our understanding of the structural components that are important for substrate specificity in this physiologically important family. We first used homology modeling, site-directed mutagenesis and transport assays to characterize the human NaDC3 (SLC13A3) and identify key residues involved in substrate and Na+ binding. We then expanded our study and generated homology models for various SLC13 transporters from the human and mouse. Virtual screening of various small molecule libraries followed by experimental testing revealed nine previously unknown inhibitors of multiple members of the familly. Ligand optimization showed that differential ligand binding specificity among SLC13 family members is largely determined by the length of the carbon chain and the size of the ligands. Additionally, we identified two key positions that define a unique binding site for each SLC13 member. Our findings rationalize substrate specificity in the SLC13 family and provide a framework for the development of more potent inhibitors.