Structure, Function and Pharmacology of SLC7 Family Members and Homologues.

Amino acids are essential components of all living cells serving as building blocks of proteins, as energy source, and as precursors of metabolites and signaling molecules. Amino acid transporters are membrane proteins that mediate the transfer of amino acids across the plasma membrane, and between compartments in cells, different cells and organs. The absence, overexpression or malfunction of specific amino acid transporters have been associated with human disease. One of the projects within the Swiss National Centre of Competence in Research (NCCR) TransCure was directed at SLC7 family amino acid transporters, with a particular focus on the heteromeric amino acid transporters 4F2hc-LAT1 (SLC3A2-SLC7A5) and 4F2hc-LAT2 (SLC3A2-SLC7A8), and the bacterial homologue AdiC. The project addressed questions of basic research (function and structure), pharmacology (identification of potent inhibitors and activators), and pre-clinical medicine (e.g., physiological role in the placenta) and disease models (e.g., tumor progression) of specific SLC7 family amino acid transporters. This review presents, summarizes and discusses selected main results obtained in this NCCR TransCure project.

Small molecule inhibitors provide insights into the relevance of LAT1 and LAT2 in materno-foetal amino acid transport.

The placenta supplies the foetus with critical nutrients such as essential amino acids (AA, eg leucine) for development and growth. It also represents a cellular barrier which is formed by a polarized, differentiated syncytiotrophoblast (STB) monolayer. Active Na+ -independent leucine transport across the placenta is mainly attributed to the System L transporters LAT1/SLC7A5 and LAT2/SLC7A8. This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009). L-leucine uptake (total dose = 167 µmol/L) was sensitive to LAT1-specific inhibition by JPH203 (EC50  = 2.55 µmol/L). The inhibition efficiency of JPH203 was increased by an additional methoxy group in the JPH203-derivate JG336 (EC50  = 1.99 µmol/L). Interestingly, JX009 showed efficient System L inhibition (EC50  = 2.35 µmol/L) and was the most potent inhibitor of leucine uptake in trophoblasts. The application of JPH203 and JX009 in Transwell® -based leucine transfer revealed LAT1 as the major accumulative transporter at the apical membrane, but other System L transporters such as LAT2 as rate-limiting for leucine efflux across the basal membrane. Therefore, differential specificity of the applied inhibitors allowed for estimation of the contribution of LAT1 and LAT2 in materno-foetal AA transfer and their potential impact in pregnancy diseases associated with impaired foetal growth.

Oligodendroglial primary cilium heterogeneity during development and demyelination/remyelination.

The primary cilium (PC) has emerged as an indispensable cellular antenna essential for signal transduction of important cell signaling pathways. The rapid acquisition of knowledge about PC biology has raised attention to PC as a therapeutic target in some neurological and psychiatric diseases. However, the role of PC in oligodendrocytes and its participation in myelination/remyelination remain poorly understood. Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes during central nervous system (CNS) development. In adult, a small percentage of OPCs remains as undifferentiated cells located sparsely in the different regions of the CNS. These cells can regenerate oligodendrocytes and participate to certain extent in remyelination. This study aims characterize PC in oligodendrocyte lineage cells during post-natal development and in a mouse model of demyelination/remyelination. We show heterogeneity in the frequency of cilium presence on OPCs, depending on culture conditions in vitro and cerebral regions in vivo during development and demyelination/remyelination. In vitro, Lithium chloride (LiCl), Forskolin and Chloral Hydrate differentially affect cilium, depending on culture environment and PC length correlates with the cell differentiation state. Beside the role of PC as a keeper of cell proliferation, our results suggest its involvement in myelination/remyelination.

Impaired Myocardial Mitochondrial Function in an Experimental Model of Anaphylactic Shock.

Anaphylactic shock (AS) is associated with a profound vasodilation and cardiac dysfunction. The cellular mechanisms underlying AS-related cardiac dysfunction are unknown. We hypothesized that myocardial mitochondrial dysfunction may be associated with AS cardiac dysfunction. In controls and sensitized Brown Norway rats, shock was induced by ovalbumin i.v bolus, and abdominal aortic blood flow (ABF), systemic mean arterial pressure (MAP), and lactatemia were measured for 15 min. Myocardial mitochondrial function was assessed with the evaluation of mitochondrial respiration, oxidative stress production by reactive oxygen species (ROS), reactive nitrogen species (RNS), and the measurement of superoxide dismutases (SODs) activity. Oxidative damage was assessed by lipid peroxidation. The mitochondrial ultrastructure was assessed using transmission electronic microscopy. AS was associated with a dramatic drop in ABF and MAP combined with a severe hyperlactatemia 15 min after shock induction. CI-linked substrate state (197 ± 21 vs. 144 ± 21 pmol/s/mg, p < 0.05), OXPHOS activity by complexes I and II (411 ± 47 vs. 246 ± 33 pmol/s/mg, p < 0.05), and OXPHOS activity through complex II (316 ± 40 vs. 203 ± 28 pmol/s/mg, p < 0.05) were significantly impaired. ROS and RNS production was not significantly increased, but SODs activity was significantly higher in the AS group (11.15 ± 1.02 vs. 15.50 ± 1.40 U/mL/mg protein, p = 0.02). Finally, cardiac lipid peroxidation was significantly increased in the AS group (8.50 ± 0.67 vs. 12.17 ± 1.44 µM/mg protein, p < 0.05). No obvious changes were observed in the mitochondrial ultrastructure between CON and AS groups. Our experimental model of AS results in rapid and deleterious hemodynamic effects and was associated with a myocardial mitochondrial dysfunction with oxidative damage and without mitochondrial ultrastructural injury.

The Evaluation of l-Tryptophan Derivatives as Inhibitors of the l-Type Amino Acid Transporter LAT1 (SLC7A5).

A series of derivatives of the substrate amino acid l-tryptophan have been investigated for inhibition of the L-type amino acid transporter LAT1 (SLC7A5), which is an emerging target in anticancer drug discovery. Of the four isomeric 4-, 5-, 6-, or 7-benzyloxy-l-tryptophans, the 5-substituted derivative was the most potent, with an IC50 of 19 µM for inhibition of [3 H]-l-leucine uptake into HT-29 human colon carcinoma cells. The replacement of the carboxy group in 5-benzyloxy-l-tryptophan by a bioisosteric tetrazole moiety led to a complete loss in potency. Likewise, the corresponding tetrazolide derived from l-tryptophan itself was found to be neither a substrate nor an inhibitor of the transporter. Increasing the steric bulk at the 5-position, while reasonably well tolerated in some cases, did not result in an improvement in potency. At the same time, none of these derivatives was found to be a substrate for LAT1-mediated transport.

The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model.

BACKGROUND: The L-type amino acid transporter 1 (LAT1/SLC7A5) transports essential amino acids across the plasma membrane. While LAT1 is overexpressed in a variety of human neoplasms, its expression and its role in thyroid cancer is currently unknown. Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy for which no effective therapy exists. The purpose of this study was to explore whether the inhibition of LAT1 in ATC would affect tumor growth both in vitro and in vivo. METHODS: LAT1 was pharmacologically blocked by JPH203 in human ATC and papillary thyroid cancer (PTC) cell lines. The effects on proliferation and mTORC1 activity were addressed in vitro. A genetically engineered mouse model of ATC was used to address the effect of blocking LAT1 on tumor growth in vivo. SLC7A5 transcription was measured in patient-derived ATC samples to address the clinical relevance of the findings. RESULTS: LAT1 block by JPH203 reduced proliferation and mTORC1 signaling in human thyroid cancer cell lines. SLC7A5 transcription was upregulated in ATC tissues derived from a genetically engineered mouse model and in ATC samples recovered from patients. JPH203 treatment induced thyroid tumor growth arrest in vivo in a fully immunocompetent mouse model of thyroid cancer. Additionally, analysis of publicly available datasets of thyroid carcinomas revealed that high LAT1 expression is associated with potentially untreatable PTC presenting reduced NIS/SLC5A5 transcription and with ATC. CONCLUSIONS: These preclinical results show that LAT1 inhibition is a novel therapeutic approach in the context of thyroid cancers, and more interestingly in untreatable thyroid cancers.

Asymmetric bromine-lithium exchange: on the importance of both the diamine ligand and the organolithium reagent.

The asymmetric bromine-lithium exchange on a series of prochiral biphenyls was investigated. As in many asymmetric organolithium reactions, the chiral ligand (here a diamine) was considered to be the enantiocontrolling element. We show here that the organolithium reagent also plays an important role.

Asymmetric bromine-lithium exchange: application toward the synthesis of natural product.

Asymmetric bromine-lithium exchange has been successfully employed to synthesize bicoumarin chiral building blocks of (+)-isokotanin A and (-)-kotanin in good yields and with an excellent level of enantioselectivity. This is the first reported example of formal syntheses, using this direct methodology, leading to the single (M)-atropoisomer of (+)-isokotanin A and (-)-kotanin building blocks, without any resolution step.