Summary of Study ST004016

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002501. The data can be accessed directly via it's Project DOI: 10.21228/M8C55X This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST004016
Study TitleEvaluation of SNAT2-dependent and sodium-dependent MeAIB uptake using GCMS
Study SummaryHY15549 cell lines expressing a dox-inducible SNAT2 cDNA and lacking endogenous Snat2 expression were plated and evaluated for MeAIB uptake by culturing cells with a gradient from 0.125-4 mM for 30-minutes in PBS for 30-minutes prior to GCMS analysis. For sodium-deficient uptake experiments, PBS was prepared using equimolar concentrations of N-methyl-D-glucamine to replace sodium chloride; potassium salts were used to replace other sodium salts. SNAT2 exhibited saturable uptake kinetics with an apparent Km of ~ were engineered to stably express a human SNAT1 cDNA or an empty vector (EV, controls) by lentiviral transduction. Either EV or +SNAT1 cell lines were plated and evaluated for MeAIB uptake by culturing cells with a gradient from 0.125-4 mM for 30-minutes in PBS for 30-minutes prior to GCMS analysis. SNAT1 exhibited saturable uptake kinetics with an apparent Km of ~0.34 mM and was significantly sodium dependent.
Institute
University of British Columbia
DepartmentBiochemistry & Molecular Biology
LaboratoryParker laboratory
Last NameParker
First NameSeth
Address950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Emailseth.parker@bcchr.ca
Phone6048753121
Submit Date2025-06-19
Raw Data AvailableYes
Raw Data File Type(s)cdf
Analysis Type DetailGC-MS
Release Date2025-07-28
Release Version1
Seth Parker Seth Parker
https://dx.doi.org/10.21228/M8C55X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002501
Project DOI:doi: 10.21228/M8C55X
Project Title:Metabolomics analysis of SNAT2-deficient cells: implications for the discovery of selective transporter inhibitors
Project Type:Manuscript
Project Summary:Amino acid uptake by the solute carrier family of transporter proteins is critical to support cell metabolism, and inhibition of transporter activity represents a tractable strategy to restrict nutrient availability to cancer cells. A small molecule inhibitor of the sodium-coupled neutral amino acid transporter 2 (SNAT2), 3-(N-methyl(4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide (MMTC/57E), was recently identified and was shown to inhibit cell proliferation when combined with glucose transport inhibitors in breast and pancreatic cancer cell lines. In this study, we use mass spectrometry-based metabolomics and establish cell-based assays for the SNAT2 transporter. We show that SNAT2 knockout cells have significant defects in amino acid availability. Using our established assays, we fail to observe that MMTC/57E inhibits SNAT2 activity likely due to its poor solubility.
Institute:University of British Columbia
Department:Biochemistry & Molecular Biology
Laboratory:Parker laboratory
Last Name:Parker
First Name:Seth
Address:950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada
Email:seth.parker@bcchr.ca
Phone:6048753121

Subject:

Subject ID:SU004154
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Female

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Factor Doxycycline (y/n) Sample source MeAIB concentration (mM)
SA461958HYiSNAT2_wDox_noNa_M125_3no Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461959HYiSNAT2_wDox_noNa_M125_r2no Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461960HYiSNAT2_wDox_noNa_M125_r1no Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461961HYiSNAT2_wDox_noNa_M250_r2no Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA461962HYiSNAT2_wDox_noNa_M250_r1no Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA461963HYiSNAT2_wDox_noNa_M250_3no Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA461964HYiSNAT2_wDox_noNa_M500_r2no Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA461965HYiSNAT2_wDox_noNa_M500_r1no Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA461966HYiSNAT2_wDox_noNa_M500_3no Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA461967HYiSNAT2_wDox_noNa_M1000_r2no Sodium yes HY15549 Pancreatic Cancer Cells 1
SA461968HYiSNAT2_wDox_noNa_M1000_r1no Sodium yes HY15549 Pancreatic Cancer Cells 1
SA461969HYiSNAT2_wDox_noNa_M1000_3no Sodium yes HY15549 Pancreatic Cancer Cells 1
SA461970HYiSNAT2_wDox_noNa_M2000_r2no Sodium yes HY15549 Pancreatic Cancer Cells 2
SA461971HYiSNAT2_wDox_noNa_M2000_r1no Sodium yes HY15549 Pancreatic Cancer Cells 2
SA461972HYiSNAT2_wDox_noNa_M2000_3no Sodium yes HY15549 Pancreatic Cancer Cells 2
SA461973HYiSNAT2_wDox_noNa_M4000_r1no Sodium yes HY15549 Pancreatic Cancer Cells 4
SA461974HYiSNAT2_wDox_noNa_M4000_r2no Sodium yes HY15549 Pancreatic Cancer Cells 4
SA461975HYiSNAT2_wDox_noNa_M4000_3no Sodium yes HY15549 Pancreatic Cancer Cells 4
SA461976HYiSNAT2_noDox_wNa_M125_r1with Sodium no HY15549 Pancreatic Cancer Cells 0.125
SA461977HYiSNAT2_noDox_wNa_M125_r2with Sodium no HY15549 Pancreatic Cancer Cells 0.125
SA461978HYiSNAT2_noDox_wNa_M125_3with Sodium no HY15549 Pancreatic Cancer Cells 0.125
SA461979HYiSNAT2_noDox_wNa_M250_r2with Sodium no HY15549 Pancreatic Cancer Cells 0.25
SA461980HYiSNAT2_noDox_wNa_M250_r1with Sodium no HY15549 Pancreatic Cancer Cells 0.25
SA461981HYiSNAT2_noDox_wNa_M250_3with Sodium no HY15549 Pancreatic Cancer Cells 0.25
SA461982HYiSNAT2_noDox_wNa_M500_r2with Sodium no HY15549 Pancreatic Cancer Cells 0.5
SA461983HYiSNAT2_noDox_wNa_M500_3with Sodium no HY15549 Pancreatic Cancer Cells 0.5
SA461984HYiSNAT2_noDox_wNa_M500_r1with Sodium no HY15549 Pancreatic Cancer Cells 0.5
SA461985HYiSNAT2_noDox_wNa_M1000_3with Sodium no HY15549 Pancreatic Cancer Cells 1
SA461986HYiSNAT2_noDox_wNa_M1000_r1with Sodium no HY15549 Pancreatic Cancer Cells 1
SA461987HYiSNAT2_noDox_wNa_M1000_r2with Sodium no HY15549 Pancreatic Cancer Cells 1
SA461988HYiSNAT2_noDox_wNa_M2000_r2with Sodium no HY15549 Pancreatic Cancer Cells 2
SA461989HYiSNAT2_noDox_wNa_M2000_r1with Sodium no HY15549 Pancreatic Cancer Cells 2
SA461990HYiSNAT2_noDox_wNa_M2000_3with Sodium no HY15549 Pancreatic Cancer Cells 2
SA461991HYiSNAT2_noDox_wNa_M4000_r1with Sodium no HY15549 Pancreatic Cancer Cells 4
SA461992HYiSNAT2_noDox_wNa_M4000_3with Sodium no HY15549 Pancreatic Cancer Cells 4
SA461993HYiSNAT2_noDox_wNa_M4000_r2with Sodium no HY15549 Pancreatic Cancer Cells 4
SA461994HYiSNAT2_wDox_wNa_M125_r2with Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461995HYiSNAT2_wDox_wNa_M125_r1with Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461996HYiSNAT2_wDox_wNa_M125_3with Sodium yes HY15549 Pancreatic Cancer Cells 0.125
SA461997HYiSNAT2_wDox_wNa_M250_r1with Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA461998HYiSNAT2_wDox_wNa_M250_r2with Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA461999HYiSNAT2_wDox_wNa_M250_3with Sodium yes HY15549 Pancreatic Cancer Cells 0.25
SA462000HYiSNAT2_wDox_wNa_M500_r2with Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA462001HYiSNAT2_wDox_wNa_M500_r1with Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA462002HYiSNAT2_wDox_wNa_M500_3with Sodium yes HY15549 Pancreatic Cancer Cells 0.5
SA462003HYiSNAT2_wDox_wNa_M1000_3with Sodium yes HY15549 Pancreatic Cancer Cells 1
SA462004HYiSNAT2_wDox_wNa_M1000_r2with Sodium yes HY15549 Pancreatic Cancer Cells 1
SA462005HYiSNAT2_wDox_wNa_M1000_r1with Sodium yes HY15549 Pancreatic Cancer Cells 1
SA462006HYiSNAT2_wDox_wNa_M2000_3with Sodium yes HY15549 Pancreatic Cancer Cells 2
SA462007HYiSNAT2_wDox_wNa_M2000_r1with Sodium yes HY15549 Pancreatic Cancer Cells 2
SA462008HYiSNAT2_wDox_wNa_M2000_r2with Sodium yes HY15549 Pancreatic Cancer Cells 2
SA462009HYiSNAT2_wDox_wNa_M4000_3with Sodium yes HY15549 Pancreatic Cancer Cells 4
SA462010HYiSNAT2_wDox_wNa_M4000_r1with Sodium yes HY15549 Pancreatic Cancer Cells 4
SA462011HYiSNAT2_wDox_wNa_M4000_r2with Sodium yes HY15549 Pancreatic Cancer Cells 4
Showing results 1 to 54 of 54

Collection:

Collection ID:CO004147
Collection Summary:Cells were washed twice with room temperature (20°C) PBS before adding 2 mL of PBS containing 0.125-4 mM MeAIB. After a 30-minute incubation at 20°C, the transport buffer was aspirated and the cells were washed three times with ice cold 0.9% NaCl. To extract metabolites, 1 mL of ice cold 80% methanol containing 2.5 nmol of 13C3-alanine was added to each well. The plates were shaken at ~500 rpm for 15 minutes at room temperature and 900 µL was transferred to a new tube and dried using a SpeedVac for GCMS analysis.
Sample Type:Tumor cells

Treatment:

Treatment ID:TR004163
Treatment Summary:No treatment.

Sample Preparation:

Sampleprep ID:SP004160
Sampleprep Summary:Dried samples were derivatized for GCMS analysis. 25 µL of methoxyamine hydrochloride (20 mg/mL, prepared in pyridine) was added to each tube, which was vortexed and incubated at 37°C for 60 minutes. Following the incubation, 25 µL of MTBSTFA + 1% TBDMSCl was added to each tube, which was vortexed and centrifuged at 21,300 x g for 15-minutes at 20°C. 40 µL of the resulting supernatant was transferred to a plastic sample vial for GCMS analysis.

Chromatography:

Chromatography ID:CH005031
Chromatography Summary:Agilent DB35-MS (30m x 0.25mm, 0.25µm) was interfaced with an inert column (5m x 0.15mm, 0 µm) to the MSD. Initial oven temperature maintained at 75°C for 3 minutes, ramped to 255°C at 7.5°C/min, held for 10 minutes, ramped to 320°C at 10 C/min, held for 5 minutes. The inlet was maintained at 270°C in splitless mode; an injection volume of 1 uL was used for all samples.
Instrument Name:Agilent 8890
Column Name:Agilent DB-35MS (30m x 0.25mm, 0.25µm) interfaced with an inert column (5m x 0.15mm, 0 µm)
Column Temperature:75°C
Flow Gradient:100% Helium
Flow Rate:1 mL/min
Solvent A:UHP Helium
Solvent B:None
Chromatography Type:GC

Analysis:

Analysis ID:AN006621
Analysis Type:MS
Chromatography ID:CH005031
Num Factors:18
Num Metabolites:4
Units:ion counts
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