An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

Dvorak, Vojtech; Wiedmer, Tabea; Inglés-Prieto, Álvaro; Altermatt, Patrick; Batoulis, Helena; Bärenz, Felix; Bender, Eckhard; Digles, Daniela; Dürrenberger, Franz; Heitman, Laura H.; IJzerman, Adriaan P.; Kell, Douglas B.; Kickinger, Stefanie; Körzö, Daniel; Leippe, Philipp; Licher, Thomas; Manolova, Vania; Rizzetto, Riccardo; Sassone, Francesca; Scarabottolo, Lia; Schlessinger, Avner; Schneider, Vanessa; Sijben, Hubert J.; Steck, Anna Lena; Sundström, Hanna; Tremolada, Sara; Wilhelm, Maria; Muelas, Marina Wright; Zindel, Diana; Steppan, Claire M.; Superti‐Furga, Giulio

doi:10.3389/fphar.2021.722889

Cited by 35 publications

(31 citation statements)

References 263 publications

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“…Despite the attractiveness of transporters being druggable targets or site-selective carriers, there is a lack of proper tools and methods to study these proteins’ biological role and usefulness in drug development [ 42 ]. Moreover, the current methods that are used for transporter studies are based on enzyme kinetics, which is not optimal [ 43 ].…”

Section: Discussionmentioning

confidence: 99%

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

et al. 2021

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l-Type amino acid transporter 1 (LAT1), expressed abundantly in the brain and placenta and overexpressed in several cancer cell types, has gained a lot of interest in drug research and development, as it can be utilized for brain-targeted drug delivery, as well as inhibiting the essential amino acid supply to cancer cells. The structure of LAT1 is today very well-known and the interactions of ligands at the binding site of LAT1 can be modeled and explained. However, less is known of LAT1′s life cycle within the cells. Moreover, the functionality of LAT1 can be measured by several different methods, which may vary between the laboratories and make the comparison of the results challenging. In the present study, the usefulness of indirect cis-inhibition methods and direct cellular uptake methods and their variations to interpret the interactions of LAT1-ligands were evaluated. Moreover, this study also highlights the importance of understanding the intracellular kinetics of LAT1-ligands, and how they can affect the regular function of LAT1 in critical tissues, such as the brain. Hence, it is discussed herein how the selected methodology influences the outcome and created knowledge of LAT1-utilizing compounds.

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Section: Discussionmentioning

confidence: 99%

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

et al. 2021

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“…Second, there is a lack of high-quality biological tools, specific, and reliable reagents and special databases. Finally, the number of functional analyses required to study such diverse objectives is still limited [ 113 ]. It is reported radioligand uptake assays have been widely employed to study LAT1 [ 114 ], but the radioligand uptake assays cannot distinguish inhibitors from substrates.…”

Section: Inhibitors Of Lat1/4f2hc and Targeted Therapymentioning

confidence: 99%

Contribution of LAT1-4F2hc in Urological Cancers via Toll-like Receptor and Other Vital Pathways

Zhao

Sakamoto

Maimaiti

et al. 2022

Cancers

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Tumor cells are known for their ability to proliferate. Nutrients are essential for rapidly growing tumor cells. In particular, essential amino acids are essential for tumor cell growth. Tumor cell growth nutrition requires the regulation of membrane transport proteins. Nutritional processes require amino acid uptake across the cell membrane. Leucine, one of the essential amino acids, has recently been found to be closely associated with cancer, which activate mTOR signaling pathway. The transport of leucine into cells requires an L-type amino acid transporter protein 1, LAT1 (SLC7A5), which requires the 4F2 cell surface antigen heavy chain (4F2hc, SLC3A2) to form a heterodimeric amino acid transporter protein complex. Recent evidence identified 4F2hc as a specific downstream target of the androgen receptor splice variant 7 (AR-V7). We stressed the importance of the LAT1-4F2hc complex as a diagnostic and therapeutic target in urological cancers in this review, which covered the recent achievements in research on the involvement of the LAT1-4F2hc complex in urinary system tumors. In addition, JPH203, which is a selective LAT1 inhibitor, has shown excellent inhibitory effects on the proliferation in a variety of tumor cells. The current phase I clinical trials of JPH203 in patients with biliary tract cancer have also achieved good results, which is the future research direction for LAT1 targeted therapy drugs.

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“…The latter is the more important one for assessing the mechanisms of drug transport [ 8 ], and there are many examples (e.g., [ 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 ]) where transporter activity has been identified but where the transporters involved have not. Of course, while a known result is a partial answer to each question, the strategies for tackling them are slightly different (for a recent overview of cell-based assay methods for SLCs, see [ 119 ]). Note, in particular, that simple biophysics plus the re-use of protein motifs in evolution means that most small molecules bind to multiple targets, and most proteins can bind small molecules promiscuously (e.g., [ 8 , 9 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 ]).…”

Section: Introductionmentioning

confidence: 99%

“…This de-orphanisation hinged upon a successful combination of genomics, metabolomics, CRISPR-Cas9-mediated gene editing, and genetics. Other strategies include binding assays [ 107 ], the use of direct assays in Xenopus oocytes [ 290 ], and others that are covered in a recent and comprehensive review [ 119 ].…”

Section: Introductionmentioning

confidence: 99%

The Transporter-Mediated Cellular Uptake and Efflux of Pharmaceutical Drugs and Biotechnology Products: How and Why Phospholipid Bilayer Transport Is Negligible in Real Biomembranes

Kell

2021

Molecules

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Over the years, my colleagues and I have come to realise that the likelihood of pharmaceutical drugs being able to diffuse through whatever unhindered phospholipid bilayer may exist in intact biological membranes in vivo is vanishingly low. This is because (i) most real biomembranes are mostly protein, not lipid, (ii) unlike purely lipid bilayers that can form transient aqueous channels, the high concentrations of proteins serve to stop such activity, (iii) natural evolution long ago selected against transport methods that just let any undesirable products enter a cell, (iv) transporters have now been identified for all kinds of molecules (even water) that were once thought not to require them, (v) many experiments show a massive variation in the uptake of drugs between different cells, tissues, and organisms, that cannot be explained if lipid bilayer transport is significant or if efflux were the only differentiator, and (vi) many experiments that manipulate the expression level of individual transporters as an independent variable demonstrate their role in drug and nutrient uptake (including in cytotoxicity or adverse drug reactions). This makes such transporters valuable both as a means of targeting drugs (not least anti-infectives) to selected cells or tissues and also as drug targets. The same considerations apply to the exploitation of substrate uptake and product efflux transporters in biotechnology. We are also beginning to recognise that transporters are more promiscuous, and antiporter activity is much more widespread, than had been realised, and that such processes are adaptive (i.e., were selected by natural evolution). The purpose of the present review is to summarise the above, and to rehearse and update readers on recent developments. These developments lead us to retain and indeed to strengthen our contention that for transmembrane pharmaceutical drug transport “phospholipid bilayer transport is negligible”.

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An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters

Cited by 35 publications

References 263 publications

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

Contribution of LAT1-4F2hc in Urological Cancers via Toll-like Receptor and Other Vital Pathways

The Transporter-Mediated Cellular Uptake and Efflux of Pharmaceutical Drugs and Biotechnology Products: How and Why Phospholipid Bilayer Transport Is Negligible in Real Biomembranes

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