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

Abstract

The glutamine transporter ASCT2 has been identified as a promising target to inhibit rapid growth of cancer cells. However, ASCT2 pharmacology is not well established. In this report, we performed a systematic structure activity analysis of a series of substituted benzylproline derivatives. Substitutions on the phenyl ring resulted in compounds with characteristics of ASCT2 inhibitors. Apparent binding affinity increased with increasing hydrophobicity of the side chain. In contrast, interaction of the ASCT2 binding site with specific positions on the phenyl ring was not observed. The most potent compound inhibits the ASCT2 anion conductance with a Ki of 3 μM, which is in the same range as that of more bulky and higher molecular weight inhibitors recently reported by others. The experimental results are consistent with computational analysis based on docking of the inhibitors against an ASCT2 homology model. The benzylproline scaffold provides a valuable tool for further improving binding potency of future ASCT2 inhibitors.

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

“…However, consistent with previous results, ASCT2 does not bind or transport lysine [1,3]. Additionally, we found that apparent affinity increased with decreasing side chain length and L-DAP binding could be inhibited competitively using previously published ASCT2 inhibitors [38]. We propose a mechanism for interaction of ASCT2 with various protonation states of L-DAP ( Figure 1D-G), based on kinetic modeling as well as molecular docking.…”

“…To investigate further if the compounds with basic side chains were actually binding to the ASCT2 binding site, we conducted competition experiments at varying concentrations of a competitive inhibitor of ASCT2, (R)-γ-(4-biphenylmethyl)-L-proline (BPP). This compound was previously characterized and competes with alanine for the substrate binding site with a K i of 3 mM [38]. Consistent with the hypothesis of L-DAP acting as a substrate, BPP reduced L-DAP induced inward currents at all concentrations and the results could be fitted with a Michaelis-Menten-like relationship to obtain the apparent binding affinity for L-DAP at three different inhibitor concentrations ( Figure 4A-C).…”

Structural basis for the transport mechanism of the human glutamine transporter SLC1A5 (ASCT2)

“…The opening of the HP2 loop increases the accessible surface area, exposing a previously unidentified cryptic pocket which could accommodate a large inhibitor that would be specific to the HP2 open state. In contrast, the closed conformation of HP2 has a small binding site, limiting the size of the inhibitors (30,31).…”

Structural basis for stereospecific inhibition of ASCT2 from rational design

“…Enrichment was done using a library of 29 known ASCT2 ligands, including substrates and inhibitors that were collected from the literature 15,16,24,[46][47][48] and ChEMBL 49 and 1,434 decoys generated with the DUD-E server 50 . Docking was performed with OpenEye FRED 51 as described in our previous work 27 .…”

“…Because ASCT2 is a pharmacologically important target, multiple small molecule competitive inhibitors have been developed for this protein, using both structure-based rational design and ligand-based approaches (reviewed in reference 22). For example, guided by homology modeling, we discovered and optimized a series of proline-based derivatives as well as other chemical scaffolds that inhibit ASCT2 in low µM potencies 16,23,24 . Notably, these amino acid analog inhibitors, which have large hydrophobic groups in the side chain provided evidence that non-polar interactions within the substrate binding site may be exploited to further increase potency.…”