#METABOLOMICS WORKBENCH Sethjparker_20250619_142348 DATATRACK_ID:6066 STUDY_ID:ST004022 ANALYSIS_ID:AN006628 PROJECT_ID:PR002501 VERSION 1 CREATED_ON July 2, 2025, 11:18 am #PROJECT PR:PROJECT_TITLE Metabolomics analysis of SNAT2-deficient cells: implications for the discovery PR:PROJECT_TITLE of selective transporter inhibitors PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY Amino acid uptake by the solute carrier family of transporter proteins is PR:PROJECT_SUMMARY critical to support cell metabolism, and inhibition of transporter activity PR:PROJECT_SUMMARY represents a tractable strategy to restrict nutrient availability to cancer PR:PROJECT_SUMMARY cells. A small molecule inhibitor of the sodium-coupled neutral amino acid PR:PROJECT_SUMMARY transporter 2 (SNAT2), PR:PROJECT_SUMMARY 3-(N-methyl(4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide PR:PROJECT_SUMMARY (MMTC/57E), was recently identified and was shown to inhibit cell proliferation PR:PROJECT_SUMMARY when combined with glucose transport inhibitors in breast and pancreatic cancer PR:PROJECT_SUMMARY cell lines. In this study, we use mass spectrometry-based metabolomics and PR:PROJECT_SUMMARY establish cell-based assays for the SNAT2 transporter. We show that SNAT2 PR:PROJECT_SUMMARY knockout cells have significant defects in amino acid availability. Using our PR:PROJECT_SUMMARY established assays, we fail to observe that MMTC/57E inhibits SNAT2 activity PR:PROJECT_SUMMARY likely due to its poor solubility. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Biochemistry & Molecular Biology PR:LABORATORY Parker laboratory PR:LAST_NAME Parker PR:FIRST_NAME Seth PR:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada PR:EMAIL seth.parker@bcchr.ca PR:PHONE 6048753121 #STUDY ST:STUDY_TITLE Evaluating competitive inhibition of SNAT2-dependent MeAIB uptake by SNAT2 ST:STUDY_TITLE knockout or treatment with L-alanine or 57E. ST:STUDY_SUMMARY MeAIB is a competitive inhibitor and transported substrate of SNAT2. In this ST:STUDY_SUMMARY study, we quantified cellular uptake of MeAIB and how L-alanine or 57E treatment ST:STUDY_SUMMARY might inhibit its uptake in HY15549 cells. ST:INSTITUTE University of British Columbia ST:DEPARTMENT Biochemistry & Molecular Biology ST:LABORATORY Parker laboratory ST:LAST_NAME Parker ST:FIRST_NAME Seth ST:ADDRESS 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada ST:EMAIL seth.parker@bcchr.ca ST:PHONE 6048753121 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 1 wDox_veh_M500_2 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=wDox_veh_M500_2.CDF SUBJECT_SAMPLE_FACTORS 2 wDox_veh_M500_1 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=wDox_veh_M500_1.CDF SUBJECT_SAMPLE_FACTORS 3 wDox_Ala20mM_M500_1 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=wDox_Ala20mM_M500_1.CDF SUBJECT_SAMPLE_FACTORS 4 wDox_57E40_M500_1 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=wDox_57E40_M500_1.CDF SUBJECT_SAMPLE_FACTORS 5 wDox_57E20_M500_1 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=wDox_57E20_M500_1.CDF SUBJECT_SAMPLE_FACTORS 6 noDox_veh_M500_2 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=noDox_veh_M500_2.CDF SUBJECT_SAMPLE_FACTORS 7 noDox_veh_M500_1 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=1; RAW_FILE_NAME(Raw file name)=noDox_veh_M500_1.CDF SUBJECT_SAMPLE_FACTORS 8 HY15549_iSNAT2_wDox_veh_M500_r3 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=3; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_veh_M500_r3.CDF SUBJECT_SAMPLE_FACTORS 9 HY15549_iSNAT2_wDox_veh_M500_r2 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=2; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_veh_M500_r2.CDF SUBJECT_SAMPLE_FACTORS 10 HY15549_iSNAT2_wDox_Ala20_M500_r3 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=3; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_Ala20_M500_r3.CDF SUBJECT_SAMPLE_FACTORS 11 HY15549_iSNAT2_wDox_Ala20_M500_r2 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=2; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_Ala20_M500_r2.CDF SUBJECT_SAMPLE_FACTORS 12 HY15549_iSNAT2_wDox_57E40_M500_r3 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=3; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_57E40_M500_r3.CDF SUBJECT_SAMPLE_FACTORS 13 HY15549_iSNAT2_wDox_57E40_M500_r2 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=2; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_57E40_M500_r2.CDF SUBJECT_SAMPLE_FACTORS 14 HY15549_iSNAT2_wDox_57E20_M500_r3 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=3; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_57E20_M500_r3.CDF SUBJECT_SAMPLE_FACTORS 15 HY15549_iSNAT2_wDox_57E20_M500_r2 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=2; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_wDox_57E20_M500_r2.CDF SUBJECT_SAMPLE_FACTORS 16 HY15549_iSNAT2_noDox_veh_M500_r3 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=3; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_noDox_veh_M500_r3.CDF SUBJECT_SAMPLE_FACTORS 17 HY15549_iSNAT2_noDox_veh_M500_r2 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Replicate=2; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_noDox_veh_M500_r2.CDF SUBJECT_SAMPLE_FACTORS 18 HY15549_iSNAT2_5DMSO_wDox_veh_M500_4 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Replicate=4; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_5DMSO_wDox_veh_M500_4.CDF SUBJECT_SAMPLE_FACTORS 19 HY15549_iSNAT2_5DMSO_wDox_Ala20_M500_4 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Replicate=4; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_5DMSO_wDox_Ala20_M500_4.CDF SUBJECT_SAMPLE_FACTORS 20 HY15549_iSNAT2_5DMSO_wDox_57E20_M500_4 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Replicate=4; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_5DMSO_wDox_57E20_M500_4.CDF SUBJECT_SAMPLE_FACTORS 21 HY15549_iSNAT2_5DMSO_noDox_veh_M500_4 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Replicate=4; RAW_FILE_NAME(Raw file name)=HY15549_iSNAT2_5DMSO_noDox_veh_M500_4.CDF #COLLECTION CO:COLLECTION_SUMMARY Cells were washed twice with room temperature (20°C) PBS before adding 2 mL of CO:COLLECTION_SUMMARY PBS containing 0.5 mM MeAIB and 20 mM of L-alanine, 20 µM of 57E, or 40 µM of CO:COLLECTION_SUMMARY 57E. A separate well was plated without doxycycline for 24-hours to withdraw CO:COLLECTION_SUMMARY SNAT2 expression (SNAT2-KO). After a 30-minute incubation at 20°C, the CO:COLLECTION_SUMMARY transport buffer was aspirated and the cells were washed three times with ice CO:COLLECTION_SUMMARY cold 0.9% NaCl. To extract metabolites, 1 mL of ice cold 80% methanol containing CO:COLLECTION_SUMMARY 2.5 nM of 13C3-alanine was added to each well. The plates were shaken at ~500 CO:COLLECTION_SUMMARY rpm for 15 minutes at room temperature and 900 µL was transferred to a new tube CO:COLLECTION_SUMMARY and dried using a SpeedVac for GCMS analysis. CO:SAMPLE_TYPE Tumor cells #TREATMENT TR:TREATMENT_SUMMARY No treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried samples were derivatized for GCMS analysis. 25 µL of methoxyamine SP:SAMPLEPREP_SUMMARY hydrochloride (20 mg/mL, prepared in pyridine) was added to each tube, which was SP:SAMPLEPREP_SUMMARY vortexed and incubated at 37°C for 60 minutes. Following the incubation, 25 µL SP:SAMPLEPREP_SUMMARY of MTBSTFA + 1% TBDMSCl was added to each tube, which was vortexed and SP:SAMPLEPREP_SUMMARY centrifuged at 21,300 x g for 15-minutes at 20°C. 40 µL of the resulting SP:SAMPLEPREP_SUMMARY supernatant was transferred to a plastic sample vial for GCMS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Agilent DB35-MS (30 m x 0.25 mm, 0.25 µm) was interfaced with an inert column CH:CHROMATOGRAPHY_SUMMARY (5 m x 0.15 mm, 0 µm) to the MSD. Initial oven temperature maintained at 75°C CH:CHROMATOGRAPHY_SUMMARY for 3 minutes, ramped to 255°C at 7.5C/min, held for 10 minutes, ramped to CH:CHROMATOGRAPHY_SUMMARY 320°C at 10 C/min, held for 5 minutes. The inlet was maintained at 270°C in CH:CHROMATOGRAPHY_SUMMARY splitless mode; an injection volume of 1 µL was used for all samples. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 8890 CH:COLUMN_NAME Agilent DB-35MS (30m x 0.25mm, 0.25µm) interfaced with an inert column (5m x 0.15mm, 0 µm) CH:SOLVENT_A UHP Helium CH:SOLVENT_B None CH:FLOW_GRADIENT 100% Helium CH:FLOW_RATE 1 mL/min CH:COLUMN_TEMPERATURE 75°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5977B MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The MS was operated in full-scan mode from 100 to 650 m/z with a threshold of MS:MS_COMMENTS 150, a scan speed of 781 u/sec, a frequency of 1.4 scans/sec, a cycle time of MS:MS_COMMENTS 723 ms, and a step size of 0.1 m/z. The MS Source and MS Quad temperatures were MS:MS_COMMENTS set to 230°C and 150°C, respectively. A gain factor of 1 was used. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples noDox_veh_M500_1 wDox_veh_M500_1 wDox_57E20_M500_1 wDox_57E40_M500_1 wDox_Ala20mM_M500_1 noDox_veh_M500_2 wDox_veh_M500_2 HY15549_iSNAT2_5DMSO_noDox_veh_M500_4 HY15549_iSNAT2_5DMSO_wDox_57E20_M500_4 HY15549_iSNAT2_5DMSO_wDox_Ala20_M500_4 HY15549_iSNAT2_5DMSO_wDox_veh_M500_4 HY15549_iSNAT2_noDox_veh_M500_r2 HY15549_iSNAT2_wDox_veh_M500_r2 HY15549_iSNAT2_wDox_Ala20_M500_r2 HY15549_iSNAT2_wDox_57E20_M500_r2 HY15549_iSNAT2_wDox_57E40_M500_r2 HY15549_iSNAT2_noDox_veh_M500_r3 HY15549_iSNAT2_wDox_veh_M500_r3 HY15549_iSNAT2_wDox_Ala20_M500_r3 HY15549_iSNAT2_wDox_57E20_M500_r3 HY15549_iSNAT2_wDox_57E40_M500_r3 Factors Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):5 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):no | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:vehicle | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:L-Alanine (20 mM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (20 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 Treatment:57E (40 microM) | Doxycycline (y/n):yes | Sample source:HY15549 Pancreatic Cancer Cells | Final DMSO (%):0.1 | MeAIB concentration (mM):0.5 MeAIB_174 1052646 11841263 11047956 12792405 1129365 629186 13067603 583341 10440710 675476 10275414 637210 7967059 691436 6493883 6603635 525776 6937604 527345 5185483 6795892 Ala_232 11085605 20259710 32486016 40041490 133023298 11209277 19854610 3031603 30199439 96561700 13008213 10798287 12686900 97079071 19854727 18411306 9318727 11091701 113251202 18129546 17772122 Ala_260 10668470 19844878 32567608 39659922 124394063 10824044 19157488 2758860 29182792 96161954 11722968 9944782 11797830 99202418 19587036 18088261 8902815 10626086 105525816 17595485 17500309 Ala_264 8693736.203 8686103.101 7516603.926 7983542.299 2985457.512 8771805.258 7678321.19 2205432.684 2611859.884 1355883.551 2364522.646 9056712.967 8960451.885 4999801.867 9893411.884 9141807.109 8052596.168 7694348.873 4432084.272 7826471.728 7710636.145 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z rt MeAIB_174 174 13.65 Ala_232 232 18.16 Ala_260 260 18.16 Ala_264 264 18.16 METABOLITES_END #END