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

Departments of Bioengineering and Therapeutic Sciences and Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, and Graduate Group in Biophysics, University of California, San Francisco, CA 94158.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2013; 110(14). DOI: 10.1073/pnas.1218165110
Source: PubMed


The Large-neutral Amino Acid Transporter 1 (LAT-1)-a sodium-independent 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.

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Available from: Jonathan E Gable, Oct 17, 2014
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    • "This may be due to the need of Leu, recognized as substrate by LAT1 (Figs. 6B and 8), during mammalian embryo development for mTOR pathway regulation; thus, absence of Leu transporter(s) is incompatible with life (Taylor, 2014). An intensive research area of investigation is represented by the transport of different drugs mediated by LAT1 (del Amo et al., 2008; Geier et al., 2013). This interest relies on LAT1 localization at the BBB which is a critical barrier for delivery of pharmacological and xenobiotic compounds to nervous tissue (Boado et al., 2005; Ylikangas et al., 2013; Zimmermann et al., 2013). "
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    ABSTRACT: LAT1 (SLC7A5) and CD98 (SLC3A2) constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. Whether one or both subunits are competent for transport is still unclear. The present work aims to solve this question using different experimental strategies. Firstly, LAT1 and CD98 were immuno-detected in protein extracts from SiHa cells. Under oxidizing conditions, i.e., without addition of SH (thiol) reducing agent DTE, both proteins were revealed as a 120 kDa major band. Upon DTE treatment separated bands, corresponding to LAT1(35 kDa) or CD98(80 kDa), were detected. LAT1 function was evaluated in intact cells as BCH sensitive [(3)H]His transport inhibited by hydrophobic amino acids. Antiport of [(3)H]His was measured in proteoliposomes reconstituted with SiHa cell extract in presence of internal His. Transport was increased by DTE. Hydrophobic amino acids were best inhibitors as well as hydrophilic Tyr, Gln, Asn and Lys. Cys, Tyr and Gln, included in the intraliposomal space, were transported in antiport with external [(3)H]His. Similar experiments were performed in proteoliposomes reconstituted with the recombinant purified hLAT1. Results overlapping those obtained with native protein were achieved. Lower transport of [(3)H]Leu and [(3)H]Gln with respect to [(3)H]His was detected. Kinetic asymmetry was found with external Km for His lower than internal one. No transport was detected in proteoliposomes reconstituted with recombinant hCD98. The experimental data demonstrate that LAT1 is the sole transport competent subunit of the heterodimer. This conclusion has important outcome for following studies on functional characterization and identification of specific inhibitors with potential application in human therapy. Copyright © 2015. Published by Elsevier Ltd.
    The international journal of biochemistry & cell biology 08/2015; 67. DOI:10.1016/j.biocel.2015.08.004 · 4.05 Impact Factor
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    • "Substrate prediction and virtual screening of drug candidates become possible with computational modeling and docking based on the available structural information. For example, novel ligands have been successfully screened from compound libraries for the norepinephrine transporter (NET, SLC6A2, Schlessinger et al., 2011), the GABA transporter 2 (GAT-2, SLC6A13, Schlessinger et al., 2012), and the largeneutral amino acid transporter (LAT-1, SLC7A5, Geier et al., 2013), based on the crystal structures of their prokaryotic homologues. Ligand discovery can also be accomplished by ligand-based modeling. "
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    ABSTRACT: Transporters comprise the largest family of membrane proteins in human organism, including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of xenobiotic and endogenous molecules. Transporters are widely expressed in various human tissues and are routinely evaluated during the process of drug development and approval. Over the past decade, increasing evidence shows that drug transporters are important in both normal physiology and disease. Currently, transporters are utilized as therapeutic targets to treat numerous diseases such as diabetes, major depression, hypertension and constipation. Despite the steady growth of the field of transporter biology, more than half of the members in transporter superfamily have little information available about their endogenous substrate(s) or physiological functions. This review outlines current research methods in transporter studies, and summarizes the drug-transporter interactions including drug-drug and drug-endogenous substrate interactions. In the end, we also discuss the therapeutic perspective of transporters based on their physiological and pathophysiological roles.
    Protein & Cell 03/2015; 6(5). DOI:10.1007/s13238-015-0148-2 · 3.25 Impact Factor
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    • "Recently, a LAT1 homology model was presented based on the crystal structure of bacterial agmatine antiporter AdiC (58). Iodotyrosines were identified in silico as LAT1 substrates and confirmed experimentally. "
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    ABSTRACT: Thyroid hormone (TH) transport into the brain is not only pivotal for development and differentiation, but also for maintenance and regulation of adult central nervous system (CNS) function. In this review, we highlight some key factors and structures regulating TH uptake and distribution. Serum TH binding proteins play a major role for the availability of TH since only free hormone concentrations may dictate cellular uptake. One of these proteins, transthyretin is also present in the cerebrospinal fluid (CSF) after being secreted by the choroid plexus. Entry routes into the brain like the blood-brain-barrier (BBB) and the blood-CSF-barrier will be explicated regarding fetal and adult status. Recently identified TH transmembrane transporters (THTT) like monocarboxylate transporter 8 (Mct8) play a major role in uptake of TH across the BBB but as well in transport between cells like astrocytes and neurons within the brain. Species differences in transporter expression will be presented and interference of TH transport by endogenous and exogenous compounds including endocrine disruptors and drugs will be discussed.
    Frontiers in Endocrinology 06/2014; 5:98. DOI:10.3389/fendo.2014.00098
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