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.

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.

Optimizing TRPM4 inhibitors in the MHFP6 chemical space.

We recently reported 4-chloro-2-(2-chlorophenoxy)acetamido)benzoic acid (CBA) as the first potent inhibitor of TRPM4, a cation channel implicated in cardiac diseases and prostate cancer. Herein we report a structure-activity relationship (SAR) study of CBA resulting in two new potent analogs. To design and interpret our SAR we used interactive color-coded 3D-maps representing similarities between compounds calculated with MHFP6 (MinHash fingerprint up to six bonds), a new molecular fingerprint outperforming other fingerprints in benchmarking virtual screening studies. We further illustrate the general applicability of our method by visualizing the structural diversity of active compounds from benchmarking sets in relation to decoy molecules and to drugs. MHFP6 chemical space 3D-maps might be generally helpful in designing, interpreting and communicating the results of SAR studies. The modified WebMolCS is accessible at http://gdb.unibe.ch and the code is available at https://github.com/reymond-group/webMolCS for off-line use.

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.

Identification of potent and selective small molecule inhibitors of the cation channel TRPM4.

BACKGROUND AND PURPOSE: TRPM4 is a calcium-activated non-selective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small-molecule inhibitors by combining in silico methods and cell-based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors. EXPERIMENTAL APPROACH: Here, we developed a high throughput screening compatible assay to record TRPM4-mediated Na+ influx in cells using a Na+ -sensitive dye and used this assay to screen a small set of compounds selected by ligand-based virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using Western blots and electrophysiology experiments. KEY RESULTS: A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss-of-expression and function, is rescued by the most promising compound 5 identified in this study. CONCLUSIONS AND IMPLICATIONS: The discovery of compound 5, a potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.