High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

Schlessinger, Avner; Wittwer, Matthias; Dahlin, Amber; Khuri, Natalia; Bonomi, Massimiliano; Fan, Hao; Giacomini, Kathleen M.; Šali, Andrej

doi:10.1074/jbc.m112.388157

Cited by 45 publications

(41 citation statements)

References 74 publications

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“…The low specificity is not surprising because the docking pipeline was executed in a fully automated fashion; in contrast, typical structure-based virtual screening involves manual postdocking selection of ligand poses. 22,23,26 We examined compounds predicted to be OCT1 ligands by the comparative model but not identified as inhibitors by HTS. In view of the published literature, we identified 13 previously reported substrates and five inhibitors of OCT1.…”

Section: ■ Resultsmentioning

confidence: 99%

“…We also performed an enrichment analysis of docked substrates and decoys and computed a logAUC metric of 22.94, which suggests docking accuracy comparable to that in previously reported virtual screening experiments for human SLC transporters. 22,23 Second, we analyzed favorable docking poses to determine the frequency of predicted hydrogen bonds between binding site residues and substrate molecules. Thirteen residues in TMHs 4, 10, and 11 formed hydrogen bonds with substrate molecules ( Figure 1A).…”

Section: ■ Resultsmentioning

confidence: 99%

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Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

et al. 2017

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Organic cation transporter 1 (OCT1) plays a critical role in the hepatocellular uptake of structurally diverse endogenous compounds and xenobiotics. Here we identified competitive and noncompetitive OCT1-interacting ligands in a library of 1780 prescription drugs by combining in silico and in vitro methods. Ligands were predicted by docking against a comparative model based on a eukaryotic homologue. In parallel, high-throughput screening (HTS) was conducted using the fluorescent probe substrate ASP+ in cells overexpressing human OCT1. Thirty competitive OCT1 ligands, defined as ligands predicted in silico as well as found by HTS, were identified. Of the 167 ligands identified by HTS, five were predicted to potentially cause clinical drug interactions. Finally, virtual screening of 29 332 metabolites predicted 146 competitive OCT1 ligands, of which an endogenous neurotoxin, 1-benzyl-1,2,3,4-tetrahydroisoquinoline, was experimentally validated. In conclusion, by combining docking and in vitro HTS, competitive and noncompetitive ligands of OCT1 can be predicted.

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Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Resultsmentioning

confidence: 99%

Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

et al. 2017

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“…Importantly, some of these newly determined structures represent different protein conformations, allowing in silico screens of small molecules against comparative models of different conformations to suggest chemically distinct ligands. For example, a structure-based approach predicted that molecules binding to a model for the outward-facing conformation of the GABA transporter 2 were chemically distinct from those predicted to bind an occluded model (44). Thus, as more structures of LAT-1 homologs are discovered, our results can be refined to identify novel LAT-1 ligands for effective therapy and the study of CNS diseases and cancer.…”

Section: Targeting Biological Systems Using a Combined Structural Phamentioning

confidence: 99%

Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1

Geier¹,

Schlessinger

Fan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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163

219

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The Large-neutral Amino Acid Transporter 1 (LAT-1)-a sodiumindependent exchanger of amino acids, thyroid hormones, and prescription drugs-is highly expressed in the blood-brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood-brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.arge-neutral Amino Acid Transporter 1 (LAT-1) is a sodiumindependent exchanger found in the brain, testis, and placenta, where it mediates transport of large-neutral amino acids (e.g., tyrosine) and thyroid hormones (e.g., triiodothyronine) across the cell membrane (1). More specifically, LAT-1 is highly expressed in the blood-and brain-facing membranes of the blood-brain barrier (BBB) to supply the central nervous system (CNS) with essential nutrients and to help maintain the neural microenvironment (2). LAT-1 is also an important drug target because it transports several prescription drugs, such as the antiparkinsonian drug L-dopa and the anticonvulsant gabapentin, across the BBB, thereby enabling their pharmacologic effects (3,4). This function at the BBB has made LAT-1 a target for drug delivery by modifying CNS-impermeable drugs such that they become LAT-1 substrates and have enhanced BBB penetration (5, 6).In addition, LAT-1 expression levels are increased in many types of cancer, including non-small-cell lung cancer and glioblastoma multiforme (GBM) (7,8). LAT-1 expression increases as cancers progress, leading to higher expression levels in highgrade tumors and metastases (9). In particular, LAT-1 plays a key role in cancer-associated reprogrammed metabolic networks by supplying growing tumor cells with essential amino acids that are used as nutrients to build biomass and signaling molecules to enhance proliferation by activating progrowth pathways such as the mammalian target of rapamycin (mTOR) pathway (10). Furthermore, inhibiting LAT-1 function reduces tumor cell proliferation, indicating that it may be a viable target for novel anticancer therapies (11-13). A cancer drug targeting LAT-1 can therefore be a LAT-1 inhibitor that deprives the cancer cells of nutrients or a cytotoxic LAT-1 substrate with an intracellular target (e.g., a metabolic enzyme).LAT-1 is a large protein with 12 putative membrane-spanning helices (14). To transport solutes across the membrane, LAT-1 binds SLC3A2, a glycoprotein with a single membrane-spanning helix that serves as a chaperone for LAT-1 (14). The atomic structure of human LAT-1 is not known, but LAT-1 exhibits significant sequence similarity to prokar...

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“…In particular, we used GROMACS4 molecular dynamics code as described previously (26). The model was subjected to 10,000 steps of conjugate gradient minimization under the Amber99SB-ILDN force field (27,28).…”

Section: Methodsmentioning

confidence: 99%

“…Molecular Docking-Docking of NaDC3 substrates was performed with DOCK 3.5.54 (30,26), which calculates scores of docking poses with van der Waals, Poisson-Boltzmann electrostatic, and substrate desolvation penalty terms. The transporter model with refined side chains was prepared by removing all non-protein atoms except for the sodium ion.…”

Section: Methodsmentioning

confidence: 99%

Determinants of Substrate and Cation Transport in the Human Na+/Dicarboxylate Cotransporter NaDC3

Schlessinger

Sun

Colas

et al. 2014

Journal of Biological Chemistry

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Background: NaDC3 transports important metabolic intermediates, including succinate and citrate, into cells. Results: We describe the NaDC3 structure and mode of substrate interaction using molecular modeling followed by experimental testing. Conclusion: The model identifies high-affinity substrate and sodium binding sites in NaDC3. Significance: The results improve our understanding of substrate binding and substrate preferences in the SLC13/DASS transporters.

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High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

Cited by 45 publications

References 74 publications

Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1

Determinants of Substrate and Cation Transport in the Human Na+/Dicarboxylate Cotransporter NaDC3

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