Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening

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.

Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2

“…Therefore, application of blockers reduces the inward leak anion current, generating apparent outward current (Fig. 3A middle panels and right panel), as reported previously for other ASCT2 blockers [11, 15–16]. In contrast, transported substrates, such as alanine, activate a substrate-dependent anion current, which in the presence of intracellular anion (SCN − ) is inward directed [5].…”

“…Among several other compounds, a proline derivative was identified, γ-2-fluorobenzyl proline, which inhibited ASCT2 with a 87μM affinity [15]. This result was surprising, because proline (Fig.…”

SLC transporters: structure, function, and drug discovery

“…For example, the aspartate transporter Glt Ph , from the Achaean organism Pyrococcus horikoshii , 25 shares sequence identity of 24%-35% with the human SLC1 family of glutamate or neutral amino acid transporters. Models of human SLC1 members including the glutamate transporter EAAT3 (SLC1A1) 26 and the neutral amino acid transporters ASCT1 (SLC1A4) 27, 28 and ASCT2 (SLC1A5) 29 based on Glt Ph structures have revealed mechanisms of ion coordination 26, 27 and differential amino acid selectivity 28 in this family; ASCT2 models were also subjected to successful ligand discovery 30 and optimization 29 campaigns.…”

“…53 This approach has been applied to discover novel ligands for a variety of challenging membrane protein targets including GPCRs 54, 55 and SLC transporters. 30, 33, 56-59 …”

“…30 For example, aminooxetane-3-carboxylate is a more efficient activator than any known natural or unnatural ASCT2 substrate. Moreover, three of these compounds were chemically different from known ASCT2 ligands (e.g., acivicin; Fig.…”