The human solute carrier (SLC) transporters are important targets for drug development.

Mutations in the SLC13A5 gene that codes for the Na(+)/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In the present study we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and treatment strategies. There are currently no effective treatments, but some anti-epileptic drugs targeting the GABA system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical anti-seizure medication decreases seizures in 4 patients. In contrast to previous reports, the ketogenic diet and fasting produce worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improve transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, our study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na(+)/citrate transporters.

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter Glt_Ph in two different conformations. Optimization of the models’ binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders. ASCT2 is a membrane protein that imports neutral amino acids into cells in exchange for intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung, where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. Recently, ASCT2 has been shown to be important for nutrient uptake in reprogrammed cancer networks. Here, we use an innovative computational approach that includes homology modeling and ligand docking to model the structure of this transporter in two distinct conformations, and virtually screen large compound libraries against these models. We use a variety of experimental assays and functional measurements to confirm seven new ligands for this transporter, including five activators and two inhibitors. This combined approach reveals specificity determinants for ligand-binding and transport, including previously unknown pockets to be targeted via structure-based drug design. The results improve our understanding of how substrate specificity is determined in amino acid transporters and provide a framework for developing potent chemical tools and potential drugs targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.