Abstract
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 GltPh 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.
Highlights
The solute carrier 1 family (SLC1) consists of five glutamate transporters (Excitatory Amino Acid Transporters, EAATs) that contribute to the regulation of synaptic concentrations of glutamate—the primary excitatory neurotransmitter in the central nervous system (CNS); and two neutral amino acid transporters (Alanine-Serine-Cysteine transporters, ASCT1 and 2) that exchange amino acids in neurons and/or cells of the peripheral tissues, to contribute to the homeostasis of intracellular concentrations of neutral amino acids [1]
The ASCT2 models in each state were optimized for protein-ligand complementarity by iteratively sampling different conformations from the initial MODELLER’s models, refining these models by sidechain modeling on a fixed backbone with SCWRL4 [26], performing minimization with molecular dynamics (MD) simulations with GROMACS [27], and evaluating how well the models can discriminate known ligands from decoys with enrichment calculations (Methods)
ASCT2 is a sodium-dependent neutral amino acid exchanger located in peripheral tissues, which is highly expressed in a variety of cancers where it provides key nutrients and signaling molecules for growth and proliferation
Summary
The solute carrier 1 family (SLC1) consists of five glutamate transporters (Excitatory Amino Acid Transporters, EAATs) that contribute to the regulation of synaptic concentrations of glutamate—the primary excitatory neurotransmitter in the central nervous system (CNS); and two neutral amino acid transporters (Alanine-Serine-Cysteine transporters, ASCT1 and 2) that exchange amino acids in neurons and/or cells of the peripheral tissues, to contribute to the homeostasis of intracellular concentrations of neutral amino acids [1]. ASCT2 (SLC1A5) is a sodium-dependent transporter located in the lung, kidney, intestines, and testis, where it transports small neutral amino acids across the cell membrane. No experimentally determined atomic structures for any of the human SLC1 family members, including ASCT2, are known. GltPh shares 24–35% sequence identity and the same number of transmembrane helices (i.e., eight) with the human SLC1 family, as well as a conserved binding site; the GltPh structure is the most suitable template for generating homology models of the SLC1 members [1,10,12]. Scopelliti et al have recently converted the substrate specificity of ASCT1 from transporting neutral amino acids to transporting glutamate by mutating three binding site amino acids (e.g., A382T), revealing previously unknown specificity determinants for the SLC1 family [15]
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