Substrate Specificity Determinants in Nutrient Solute Carrier Transporters

总结

SUMMARY Alterations in cell metabolism support rapid growth and proliferation of cells in pathologies such as cancer, au- toimmune disease, and heart ischemia, resulting in increased reliance on the metabolism of amino acids such as glutamine and leucine. Nutrient Solute Carrier (SLC) transporters play a major role in reprogrammed meta- bolic networks by supplying cells with nutrients that are used to build biomass, serve as signaling molecules that enhance cell proliferation and differentiation, or regulate cell death. Our broad goal is to describe the sub- strate and inhibitor specificity determinants in disease-related nutrient SLC transporters and develop unique strategies to modulate their functions. We take an integrative approach that includes computational chemistry methods, coupled with biochemical and biophysical approaches and disease-related cell lines, to char- acterize two amino acid transporters that play a key role in metabolism of rapidly-growing cells: the Alanine- Serine-Cysteine Transporter (SLC1A5, ASCT2), a Na+-dependent amino acid exchanger that modulates intra- cellular glutamine levels, and the Amino Acid Transporter B0+ (ATB0+, SLC6A14), a neutral and cationic amino acid transporter, driven by Na+ and Cl- co-transport. In Aim 1 of this project, we will continue characterizing ASCT2, a well-validated drug target for various patholo- gies (eg triple negative breast cancer and prostate cancer). Despite recent advancements in our understanding of ASCT2 structure and function made by us and others, many aspects of its biology are highly unexplored. We will rationally design chemical tools that modulate the activity of ASCT2 using unique mechanisms, including: (A) allosteric inhibitors interacting with a recently identified allosteric site; (B) covalent inhibitors targeting a unique cysteine residue in the substrate binding site of ASCT2; and (C) conformation-specific small molecule modulators targeting specific subpockets in the substrate binding site. In Aim 2, we will characterize SLC6A14, an understudied transporter involved in cancer and metabolic diseases. We will develop structural models of SLC6A14 in different conformations. We will describe biophysical features of the models’ substrate binding site, including electrostatic potential, size, shape, and hydrophobicity, to develop hypotheses for the substrate and inhibitor specificity determinants in SLC6A14. We will use this knowledge to guide the development of inhibitors and substrates, including photoactivatable compounds, to directly test inhibitor-binding site interaction(s). Successful completion of this project will provide a greater understanding of mechanisms of transport and inhi- bition of nutrient transporters, as well as novel chemical tools to further characterize their role in disease. Notably, we will test an emerging and innovative approach to transporter drug discovery that targets allosteric modulation and covalent inhibition via small molecules, to deprive hyper-proliferating cells of nutrients, potentially expanding future applications to treat other diseases that involve SLCs.