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

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid trans- porter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glu- tamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting spe- cific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final struc- tures combined with molecular dynamics simulations reveal multi- ple 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 frame- work for future development of cancer therapeutics targeting nu- trient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.