title: Development of a cell-based screening method for compounds that inhibit or are transported by large neutral amino acid transporter 1, a key transporter at the blood-brain barrier.
abstract: Large neutral amino acid transporter 1 (LAT1) transports neutral amino acids with aromatic or branched side chains as well as their derivatives or prodrugs. Because the transporter is highly expressed at the blood-brain barrier and in some tumor cells, it is a potential target to treat brain disease and cancer. Therefore, it is essential to develop a method to screen for LAT1 inhibitors or for therapeutic compounds that it can transport. In this study, one such method was developed that combines an in vitro cell-based assay with high-throughput ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QQQ-MS). Using this method, candidate compounds could be tested for the ability to inhibit or to compete with uptake of gabapentin, an LAT1 substrate, in HT-29 cells, which abundantly express the transporter. Gabapentin uptake is measured by mass spectrometry, which requires as little as 6min/sample and will enable analysis of large numbers of samples. We anticipate that the method will be useful to identify LAT1 inhibitors or substrates without the need for animals or radioactive labeling.
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, 200240 Shanghai, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Instrumental Analysis Center, Shanghai Jiao Tong University, 200240 Shanghai, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Liang
authors.affiliation: Instrumental Analysis Center, Shanghai Jiao Tong University, 200240 Shanghai, China.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Instrumental Analysis Center, Shanghai Jiao Tong University, 200240 Shanghai, China. Electronic address: lizhang09@sjtu.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
mesh_terms: D000588:Amines / Q000378:metabolism* / Q000494:pharmacology*; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism* / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D000077206:Gabapentin; D019073:HT29 Cells; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000378:metabolism*; D013058:Mass Spectrometry; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D000588:Amines; D003509:Cyclohexanecarboxylic Acids; D027283:Large Neutral Amino Acid-Transporter 1; D005680:gamma-Aminobutyric Acid; D000077206:Gabapentin
keywords: Blood–brain barrier; Cell-based assay; Gabapentin; LAT1; Mass spectrometry; Transporter