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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    • "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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    ABSTRACT: The primary objective of this study is to functionally characterize and provide molecular evidence of large neutral amino acid transporter (LAT1) in human derived prostate cancer cells (PC-3). We carried out the uptake of [3H]-tyrosine to assess the functional activity of LAT1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis is carried out to confirm the molecular expression of LAT1. [3H]-tyrosine uptake is found to be time dependent and linear up to 60min. The uptake process does not exhibit any dependence on sodium ions, pH and energy. However, it is temperature dependent and found maximal at physiological temperature. The uptake of [3H]-tyrosine demonstrates saturable kinetics with K(m) and V(max) values of 34±3μM and 0.70±0.02 nanomoles/min/mg protein, respectively. It is strongly inhibited by large neutral (phenylalanine, tryptophan, leucine, isoleucine) and small neutral (alanine, serine, cysteine) but not by basic (lysine and arginine) and acidic (aspartic and glutamic acid) amino acids. Isoleucine-quinidine (Ile-quinidine) prodrug generates a significant inhibitory effect on [3H]-tyrosine uptake suggesting that it is recognized by LAT1. RT-PCR analysis provided a product band at 658 and 840bp, specific to LAT1 and LAT2, respectively. For the first time, this study demonstrates that LAT1, primarily responsible for the uptake of large neutral amino acids, is functionally active in PC-3 cells. Significant increase in the uptake generated by Ile-quinidine relative to quinidine suggests that LAT1 can be utilized for enhancing the cellular permeation of poor cell permeable anticancer drugs. Furthermore, this cell line can be utilized as an excellent in vitro model for studying the interaction of large neutral amino acid conjugated drugs with LAT1 transporter.
    International Journal of Pharmaceutics 12/2012; 443(1). DOI:10.1016/j.ijpharm.2012.12.029 · 3.65 Impact Factor
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