Structure-Based Identification of Inhibitors for the SLC13 Family of Na⁺/Dicarboxylate Cotransporters

In mammals, citric acid cycle intermediates play a key role in regulating various metabolic processes, such as fatty acid synthesis and glycolysis. Members of the sodium-dependent SLC13 transporter family mediate the transport of di- and tricarboxylates into cells. SLC13 family members have been implicated in lifespan extension and resistance to high-fat diets; thus, they are emerging drug targets for aging and metabolic disorders. We previously characterized key structural determinants of substrate and cation binding for the human NaDC3/SLC13A3 transporter using a homology model. Here, we combine computational modeling and virtual screening with functional and biochemical testing, to identify nine previously unknown inhibitors for multiple members of the SLC13 family from human and mouse. Our results reveal previously unknown substrate selectivity determinants for the SLC13 family, including key residues that mediate ligand binding and transport, as well as promiscuous and specific SLC13 small molecule ligands. The newly discovered ligands can serve as chemical tools for further characterization of the SLC13 family or as lead molecules for the future development of potent inhibitors for the treatment of metabolic diseases and aging. Our results improve our understanding of the structural components that are important for substrate specificity in this physiologically important family as well as in other structurally related transport systems

Mapping Functionally Important Residues in the Na⁺/Dicarboxylate Cotransporter, NaDC1

Transporters from the SLC13 family couple the transport of two to four Na⁺ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na⁺/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na⁺ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased <i>K</i>_m values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na⁺. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters

Chemical Modulation of the Human Oligopeptide Transporter 1, hPepT1

In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target

Table_1_Homology Modeling Informs Ligand Discovery for the Glutamine Transporter ASCT2.DOCX

<p>The Alanine-Serine-Cysteine transporter (SLC1A5, ASCT2), is a neutral amino acid exchanger involved in the intracellular homeostasis of amino acids in peripheral tissues. Given its role in supplying glutamine to rapidly proliferating cancer cells in several tumor types such as triple-negative breast cancer and melanoma, ASCT2 has been identified as a key drug target. Here we use a range of computational methods, including homology modeling and ligand docking, in combination with cell-based assays, to develop hypotheses for structure-function relationships in ASCT2. We perform a phylogenetic analysis of the SLC1 family and its prokaryotic homologs to develop a useful multiple sequence alignment for this protein family. We then generate homology models of ASCT2 in two different conformations, based on the human EAAT1 structures. Using ligand enrichment calculations, the ASCT2 models are then compared to crystal structures of various homologs for their utility in discovering ASCT2 inhibitors. We use virtual screening, cellular uptake and electrophysiology experiments to identify a non-amino acid ASCT2 inhibitor that is predicted to interact with the ASCT2 substrate binding site. Our results provide insights into the structural basis of substrate specificity in the SLC1 family, as well as a framework for the design of future selective and potent ASCT2 inhibitors as cancer therapeutics.</p

Image_1_Homology Modeling Informs Ligand Discovery for the Glutamine Transporter ASCT2.pdf

<p>The Alanine-Serine-Cysteine transporter (SLC1A5, ASCT2), is a neutral amino acid exchanger involved in the intracellular homeostasis of amino acids in peripheral tissues. Given its role in supplying glutamine to rapidly proliferating cancer cells in several tumor types such as triple-negative breast cancer and melanoma, ASCT2 has been identified as a key drug target. Here we use a range of computational methods, including homology modeling and ligand docking, in combination with cell-based assays, to develop hypotheses for structure-function relationships in ASCT2. We perform a phylogenetic analysis of the SLC1 family and its prokaryotic homologs to develop a useful multiple sequence alignment for this protein family. We then generate homology models of ASCT2 in two different conformations, based on the human EAAT1 structures. Using ligand enrichment calculations, the ASCT2 models are then compared to crystal structures of various homologs for their utility in discovering ASCT2 inhibitors. We use virtual screening, cellular uptake and electrophysiology experiments to identify a non-amino acid ASCT2 inhibitor that is predicted to interact with the ASCT2 substrate binding site. Our results provide insights into the structural basis of substrate specificity in the SLC1 family, as well as a framework for the design of future selective and potent ASCT2 inhibitors as cancer therapeutics.</p

Image_2_Homology Modeling Informs Ligand Discovery for the Glutamine Transporter ASCT2.PDF

<p>The Alanine-Serine-Cysteine transporter (SLC1A5, ASCT2), is a neutral amino acid exchanger involved in the intracellular homeostasis of amino acids in peripheral tissues. Given its role in supplying glutamine to rapidly proliferating cancer cells in several tumor types such as triple-negative breast cancer and melanoma, ASCT2 has been identified as a key drug target. Here we use a range of computational methods, including homology modeling and ligand docking, in combination with cell-based assays, to develop hypotheses for structure-function relationships in ASCT2. We perform a phylogenetic analysis of the SLC1 family and its prokaryotic homologs to develop a useful multiple sequence alignment for this protein family. We then generate homology models of ASCT2 in two different conformations, based on the human EAAT1 structures. Using ligand enrichment calculations, the ASCT2 models are then compared to crystal structures of various homologs for their utility in discovering ASCT2 inhibitors. We use virtual screening, cellular uptake and electrophysiology experiments to identify a non-amino acid ASCT2 inhibitor that is predicted to interact with the ASCT2 substrate binding site. Our results provide insights into the structural basis of substrate specificity in the SLC1 family, as well as a framework for the design of future selective and potent ASCT2 inhibitors as cancer therapeutics.</p

Chloroalanine, AOC and γ-FBP inhibit ASCT2-mediated glutamine uptake and cell viability in C8161 human melanoma cells.

<p>(<b>A</b>-<b>C</b>) [³H]-L-glutamine uptake in C8161 cells was used to determine the IC₅₀ of chloroalanine, AOC and <b>γ</b>-FBP (n = 3). (<b>D</b>-<b>F</b>) MTT cell viability assay (n = 3) in C8161 cells incubated with chloroalanine (5 mM), AOC (5 mM) and <b>γ</b>-FBP (5 mM). Two-way ANOVA test was performed to determine significance. (<b>G</b>-<b>I</b>) Apoptosis (Early, Annexin V+ PI-; Late, Annexin V+PI+) was examined by flow cytometry in C8161 cells incubated with chloroalanine (5 mM), AOC (5 mM) and <b>γ</b>-FBP (5 mM). Mann Whitney U test was used to determine significance. (J) ASCT2 expression (with GAPDH as a loading control) in C8161 cells was assessed after 48 hours incubation with chloroalanine (5 mM), AOC (5 mM) and <b>γ</b>-FBP (5 mM).</p

Electrophysiological methods confirm predicted activators and inhibitors.

<p>(<b>A</b>-<b>C</b>) Representative whole-cell current traces in response to 1 mM of alanine, L-DOPS, and aminooxetane-3-carboxylate (AOC) applied at the time indicated by the gray bar. (<b>D</b>) Dose response curves for AOC and penicillamine (membrane potential = 0 mV, internal buffer contained 130 mM NaSCN and 10 mM alanine, external buffer contained 140 mM NaCl). (<b>E</b>) Maximum whole-cell currents relative to that induced by 1 mM alanine (membrane potential = 0 mV, internal buffer contained 130 mM NaSCN and 10 mM alanine, external buffer contained 140 mM NaCl).</p

ASCT2 models in different conformations reveal gating mechanism of the HP2 loop.

<p>Side (<b>A</b>) and cytoplasmic (<b>B</b>) view of the ASCT2 models in the occluded conformation are represented in gray ribbons. The HP2 loop of the outward-open conformation (pink ribbons) is superposed to the occluded model. Atoms of the substrate cysteine are shown as spheres where oxygen atoms are displayed in red, sulfur in yellow, and carbon atoms in cyan. Sodium ions are illustrated as small purple spheres.</p

Identification of Cinnamic Acid Derivatives As Novel Antagonists of the Prokaryotic Proton-Gated Ion Channel GLIC

Pentameric ligand gated ion channels (pLGICs) mediate signal transduction. The binding of an extracellular ligand is coupled to the transmembrane channel opening. So far, all known agonists bind at the interface between subunits in a topologically conserved “orthosteric site” whose amino acid composition defines the pharmacological specificity of pLGIC subtypes. A striking exception is the bacterial proton-activated GLIC protein, exhibiting an uncommon orthosteric binding site in terms of sequence and local architecture. Among a library of Gloeobacter violaceus metabolites, we identified a series of cinnamic acid derivatives, which antagonize the GLIC proton-elicited response. Structure–activity analysis shows a key contribution of the carboxylate moiety to GLIC inhibition. Molecular docking coupled to site-directed mutagenesis support that the binding pocket is located below the classical orthosteric site. These antagonists provide new tools to modulate conformation of GLIC, currently used as a prototypic pLGIC, and opens new avenues to study the signal transduction mechanism