Structural properties for selective and efficient l-type amino acid transporter 1 (LAT1) mediated cellular uptake

Abstract

l-Type amino acid transporter 1 (LAT1) is a sodium-independent exchanger transporting large neural amino acids and several amino-acid mimicking drugs across the cell membranes. LAT1 is highly expressed at the blood brain barrier (BBB) and in numerous cancer cells and is therefore a potential drug target. However, structural features affecting the ability to bind to LAT1 and the cellular translocation by LAT1 are unclear. In the present study we determined the binding to and transport through human LAT1 of several compounds into the human breast adenocarcinoma cells (MCF-7). We show that the meta-conjugation of l-phenylalanine increases binding to human LAT1 compared to para-conjugation or aliphatic amino acid moiety. Furthermore, large, rigid and aromatic meta-substituted l-phenylalanine derivative enabled selective and efficient LAT1-mediated cellular uptake. Our results also demonstrates that in addition to binding studies, it is of utmost importance to determine the cellular accumulation of compounds. It provides crucial information on transport efficiency and selectivity of transport mechanisms that the compounds are able to utilize. Overall, these structural findings and the methodology used herein are exploitable to design LAT1-utilizing compounds, such as markers for cancer imaging and drug molecules, enabling more effective and safer treatments for cancer in the future.

Introduction

l-type amino acid transporter 1 (LAT1) belongs to the l-type amino acid transporter family and is a sodium-independent, heterodimeric transmembrane exchanger (Kanai et al., 1998, Yanagida et al., 2001). LAT1 transports across the cell membranes large neutral amino acids such as l-leucine, l-phenylalanine and l-tryptophan, as well as several amino acid-mimicking drugs including levodopa, gabapentin and melphalan (Cornford et al., 1992, Dickens et al., 2013, Kageyama et al., 2000, Kanai et al., 1998, Yanagida et al., 2001). LAT1 is found mostly in the brain, testis and placenta (Boado et al., 1999, Kanai et al., 1998, Yanagida et al., 2001). More specifically, LAT1 is highly expressed at the blood brain barrier (BBB). LAT1 is also overexpressed in various cancer cells, including breast cancer, non-small cell lung cancer and glioblastoma multiforme (Furuya et al., 2012, Kaira et al., 2008a, Kobayashi et al., 2008). The functional level of LAT1 correlates with cancer progression; metastases and high grade tumors express greater amounts of LAT1 (Furuya et al., 2012, Kaira et al., 2008b, Papin-Michault et al., 2016). Moreover, cancer cell proliferation decreases when the function of the LAT1 is inhibited (Huttunen et al., 2016b, Oda et al., 2010, Shennan and Thomson, 2008). Therefore, LAT1 is an interesting drug target: it can be utilized as a diagnostic marker for cancer imaging; as a therapeutic target to starve cancer cells by inhibiting its function; or as a delivery mechanism for carrier-mediated transport of drugs to cancer cells or through the BBB to the central nervous system (CNS) (Jin et al., 2015, Rautio et al., 2013).

A molecule utilizing LAT1 may mimic a natural substrate of LAT1, as do the small size compounds gabapentin and levodopa, or constitute a part of larger molecule containing structural features for LAT1 recognition (Gynther et al., 2010, Gynther et al., 2016, Huttunen et al., 2016b, Peura et al., 2011, Peura et al., 2013, Puris et al., 2017). Several prodrugs of such type with various amino acid promoieties have been demonstrated to utilize LAT1 for their BBB-permeation in in situ rat brain perfusion experiments or for cellular accumulation in in vitro studies, or for both. However, while structural properties responsible for efficient LAT1 affinity have been successfully characterized (Ylikangas et al., 2013, Ylikangas et al., 2014), properties affecting the binding to LAT1 and the cellular translocation by LAT1 are not comprehensively understood. In addition, amino acid derivatives binding to LAT1 may also be substrates for other amino acid transporters, such as monocarboxylate transporters 8 and 10 (MCT8 and MCT10) (Kinne et al., 2011) or organic anions transporting polypeptides (OATPs) (Huttunen et al., 2016b), which can complicate targeted drug delivery via LAT1, and e.g. impair the selective delivery into the cancer cells.

In this study, our aim was to characterize systematically the structural features for efficient and selective binding to and transport through human LAT1. Knowlegde of these features enables more rational design of LAT1-targeted compounds through better understanding of structure-activity relationship of a transporter and a ligand/substrate. The efficient LAT1-mediated disposition and high selectivity of compounds for LAT1 over other amino acid transporters is of great importance for the success of the transporter mediated drug targeting approach. In order to achieve our aim we evaluated the ability of ten selected compounds to bind to human LAT1 (Table 1) in a LAT1-expressing cell line (human breast adenocarcinoma cancer cell line; MCF-7). Furthermore, we studied LAT1-selective translocation and uptake of compounds into these cells. The selected compounds included clinically used LAT1-utilizing drugs, investigational amino acid derivatives and natural LAT1 substrates. LAT1-selectivity was determined over two other amino acid transporters, l-type amino acid transporter 2 (LAT2) and alanine-serine-cysteine transporter 2 (ASCT2). ASCT2 was selected for the selectivity studies because this transporter is overexpressed in cancerous cells (Fuchs and Bode, 2005, Wise and Thompson, 2010) sharing mutual ligands with LAT1 (Colas et al., 2015). LAT2 was selected beacause it has overlapping substrate specificity with LAT1, but it exists ubiquitously in normal tissues (Pineda et al., 1999, Rossier et al., 1999, Segawa et al., 1999). The results of this study contribute to the current knowledge of efficient and selective LAT1-mediated uptake of compounds, facilitating more rational design of compounds utilizing LAT1 to reach CNS or selectively to target cancer cells overexpressing LAT1.