The Na+/citrate transporter, NaCT (SLC13A5), is a therapeutic target for metabolic diseases. Citrate is an important signaling molecule that regulates the activity of lipid- and glucose-metabolizing enzymes in cells. Previous studies identified two compounds, PF-06649298 (compound 2: ) and PF-06678419 (compound 4: ), that inhibit human NaCT with high affinity, and one of the compounds demonstrated specificity relative to other SLC13 family members. Here we use molecular modeling and site-directed mutagenesis of hNaCT followed by transport characterization and cell-surface biotinylation to examine the residues involved in inhibitor binding and transport. The results indicate that residues located near the putative citrate binding site, G228, V231, V232, and G409, affect both citrate transport and inhibition of citrate uptake by compounds 2: and 4: V231 appears to distinguish between compounds 2: and 4: as inhibitors. Furthermore, residues located outside of the putative citrate binding site, Q77 and T86, may also play a role in NaCT inhibition by compounds 2: and 4: Our results provide new insight into the mechanism of transport and inhibition in NaCT and the SLC13 family. These findings should provide a basis for future drug design of SLC13 inhibitors.
Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.