Electrophysiological characterization of the inhibitory effects of 2-amino-4-bis (aryloxy benzyl) amino butanoic acids on alanine serine cysteine transporter 2 and sodium coupled neutral amino acid transporter 2

Alanine Serine Cysteine Transporter 2 (ASCT2) is a member of the solute carrier 1A (SLC1A) family of transport proteins. It is a Na+ dependent, obligatory neutral amino acid exchanger with the capability to transport glutamine and it is a primary glutamine transport protein in cancer cells. This glutamine transport capability confers an important physiological role for ASCT2 in the maintenance of intracellular amino acid pools for various metabolic and cell signaling pathways. Because of this, ASCT2 is found to be overexpressed in many cancers, which rely heavily on glutamine as an essential nutrient for cell survival and growth. A wealth of research demonstrating overexpression of ASCT2 in cancer cells has made it a target for the synthesis of specific pharmacological inhibitors with the goal of killing cancer cells by glutamine starvation. A recent class of compounds known as AABA (2-amino-4-bis (aryloxy benzyl) amino butanoic acids) have been reported to have high binding affinity to ASCT2 and inhibit its function. The specificity of these inhibitors was contested and it was found that two of the most potent compounds synthesized, Compound 12 and V-9302, do not bind to ASCT2, but instead inhibit the function of two membrane transporters of different families, SNAT2 and LAT1. However, electrophysiological characterization of these inhibitors to either ASCT2 or SNAT2 has not been reported in the literature. Electrophysiological characterization has several benefits, including its high time resolution and accurate current recordings of membrane transport processes, that make it useful for measuring kinetic parameters associated with substrate or inhibitor binding such as Ki values. Herein we present the first electrophysiological characterization of several AABA compounds towards ASCT2 and SNAT2 by whole cell patch clamp current recordings and illustrate that each tested compound show variability in their inhibition toward ASCT2 or SNAT2, with some compounds presenting as more potent inhibitors of ASCT2 while others as more potent inhibitors of SNAT2. However, each compound was shown to bind to and inhibit the protein in some manner, some, albeit, at much higher concentrations than reported in the literature. Notably some of the tested compounds are the first known blockers of SNAT2 transport

Rational design of ASCT2 inhibitors using an integrated experimental-computational approach

International audienceASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery