#METABOLOMICS WORKBENCH Sethjparker_20250619_124445 DATATRACK_ID:6062 STUDY_ID:ST004014 ANALYSIS_ID:AN006619 PROJECT_ID:PR002501 VERSION 1 CREATED_ON June 30, 2025, 10:36 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 Competitive inhibition of SNAT2-dependent MeAIB uptake by saturating ST:STUDY_TITLE concentrations of L-alanine. ST:STUDY_SUMMARY MeAIB uptake in SNAT2-expressing HY15549 cells was inhibited by co-treating with ST:STUDY_SUMMARY a concentration gradient of L-alanine from 20 mM to 0.04 mM and 0.5 mM of MeAIB. 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 A0_r1 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=A0_r1.CDF SUBJECT_SAMPLE_FACTORS 2 A0_r2 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=A0_r2.CDF SUBJECT_SAMPLE_FACTORS 3 A1 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=20 mM; RAW_FILE_NAME(Raw file name)=A1.CDF SUBJECT_SAMPLE_FACTORS 4 A2 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=10 mM; RAW_FILE_NAME(Raw file name)=A2.CDF SUBJECT_SAMPLE_FACTORS 5 A3 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=5 mM; RAW_FILE_NAME(Raw file name)=A3.CDF SUBJECT_SAMPLE_FACTORS 6 A4 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=2.5 mM; RAW_FILE_NAME(Raw file name)=A4.CDF SUBJECT_SAMPLE_FACTORS 7 A5 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=1.25 mM; RAW_FILE_NAME(Raw file name)=A5.CDF SUBJECT_SAMPLE_FACTORS 8 A6 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.625 mM; RAW_FILE_NAME(Raw file name)=A6.CDF SUBJECT_SAMPLE_FACTORS 9 A7 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.3125 mM; RAW_FILE_NAME(Raw file name)=A7.CDF SUBJECT_SAMPLE_FACTORS 10 A8 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.156 mM; RAW_FILE_NAME(Raw file name)=A8.CDF SUBJECT_SAMPLE_FACTORS 11 A9 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.078 mM; RAW_FILE_NAME(Raw file name)=A9.CDF SUBJECT_SAMPLE_FACTORS 12 A10 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.039 mM; RAW_FILE_NAME(Raw file name)=A10.CDF SUBJECT_SAMPLE_FACTORS 13 B0_r1 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=B0_r1.CDF SUBJECT_SAMPLE_FACTORS 14 B0_r2 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=B0_r2.CDF SUBJECT_SAMPLE_FACTORS 15 B1 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=20 mM; RAW_FILE_NAME(Raw file name)=B1.CDF SUBJECT_SAMPLE_FACTORS 16 B2 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=10 mM; RAW_FILE_NAME(Raw file name)=B2.CDF SUBJECT_SAMPLE_FACTORS 17 B3 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=5 mM; RAW_FILE_NAME(Raw file name)=B3.CDF SUBJECT_SAMPLE_FACTORS 18 B4 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=2.5 mM; RAW_FILE_NAME(Raw file name)=B4.CDF SUBJECT_SAMPLE_FACTORS 19 B5 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=1.25 mM; RAW_FILE_NAME(Raw file name)=B5.CDF SUBJECT_SAMPLE_FACTORS 20 B6 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.625 mM; RAW_FILE_NAME(Raw file name)=B6.CDF SUBJECT_SAMPLE_FACTORS 21 B7 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.3125 mM; RAW_FILE_NAME(Raw file name)=B7.CDF SUBJECT_SAMPLE_FACTORS 22 B8 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.156 mM; RAW_FILE_NAME(Raw file name)=B8.CDF SUBJECT_SAMPLE_FACTORS 23 B9 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.078 mM; RAW_FILE_NAME(Raw file name)=B9.CDF SUBJECT_SAMPLE_FACTORS 24 B10 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.039 mM; RAW_FILE_NAME(Raw file name)=B10.CDF SUBJECT_SAMPLE_FACTORS 25 C0_r1 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=C0_r1.CDF SUBJECT_SAMPLE_FACTORS 26 C0_r2 Factor:Untreated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0 mM; RAW_FILE_NAME(Raw file name)=C0_r2.CDF SUBJECT_SAMPLE_FACTORS 27 C1 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=20 mM; RAW_FILE_NAME(Raw file name)=C1.CDF SUBJECT_SAMPLE_FACTORS 28 C2 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=10 mM; RAW_FILE_NAME(Raw file name)=C2.CDF SUBJECT_SAMPLE_FACTORS 29 C3 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=5 mM; RAW_FILE_NAME(Raw file name)=C3.CDF SUBJECT_SAMPLE_FACTORS 30 C4 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=2.5 mM; RAW_FILE_NAME(Raw file name)=C4.CDF SUBJECT_SAMPLE_FACTORS 31 C5 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=1.25 mM; RAW_FILE_NAME(Raw file name)=C5.CDF SUBJECT_SAMPLE_FACTORS 32 C6 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.625 mM; RAW_FILE_NAME(Raw file name)=C6.CDF SUBJECT_SAMPLE_FACTORS 33 C7 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.3125 mM; RAW_FILE_NAME(Raw file name)=C7.CDF SUBJECT_SAMPLE_FACTORS 34 C8 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.156 mM; RAW_FILE_NAME(Raw file name)=C8.CDF SUBJECT_SAMPLE_FACTORS 35 C9 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.078 mM; RAW_FILE_NAME(Raw file name)=C9.CDF SUBJECT_SAMPLE_FACTORS 36 C10 Factor:Treated | Sample source:HY15549 MeAIB concentration=0.5 mM; L-alanine concentration=0.039 mM; RAW_FILE_NAME(Raw file name)=C10.CDF #COLLECTION CO:COLLECTION_SUMMARY Cells were washed twice with room temperature (20oC) PBS before adding 2 mL of CO:COLLECTION_SUMMARY PBS containing 0.5 mM MeAIB and 0-20 mM of L-alanine. After a 30-minute CO:COLLECTION_SUMMARY incubation at 20oC, the transport buffer was aspirated and the cells were washed CO:COLLECTION_SUMMARY three times with ice cold 0.9% NaCl. To extract metabolites, 1 mL of ice cold CO:COLLECTION_SUMMARY 80% methanol containing 2.5 nmol of 13C3-alanine was added to each well. The CO:COLLECTION_SUMMARY plates were shaken at ~500 rpm for 15 minutes at room temperature and 900 uL was CO:COLLECTION_SUMMARY transferred to a new tube 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 uL 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 37oC for 60 minutes. Following the incubation, 25 uL SP:SAMPLEPREP_SUMMARY of MTBSTFA + 1% TBDMSCl was added to each tube, which was vortexed and SP:SAMPLEPREP_SUMMARY centrifuged at 21,300 xg for 15-minutes at 20oC. 40 uL of the resulting SP:SAMPLEPREP_SUMMARY supernatant was transferred to a plastic sample vial for GCMS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Agilent DB35-MS (30m x 0.25mm, 0.25um) was interfaced with an inert column (5m x CH:CHROMATOGRAPHY_SUMMARY 0.15mm, 0 um) to the MSD. Initial oven temperature maintained at 75C for 3 CH:CHROMATOGRAPHY_SUMMARY minutes, ramped to 255C at 7.5C/min, held for 10 minutes, ramped to 320C at 10 CH:CHROMATOGRAPHY_SUMMARY C/min, held for 5 minutes. The inlet was maintained at 270C in splitless mode; CH:CHROMATOGRAPHY_SUMMARY an injection volume of 1 ul was used for all samples. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 8890 CH:COLUMN_NAME Agilent DB-35MS CH:SOLVENT_A UHP Helium CH:SOLVENT_B None CH:FLOW_GRADIENT 100% A CH:FLOW_RATE 1 mL/min CH:COLUMN_TEMPERATURE 75 CH:INJECTION_TEMPERATURE 270 CH:OVEN_TEMPERATURE 75C #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 230C and 150C, respectively. A gain factor of 1 was used. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples A0_r1 A0_r2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 B0_r1 B0_r2 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 C0_r1 C0_r2 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Factors Factor:Untreated | Sample source:HY15549 Factor:Untreated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Untreated | Sample source:HY15549 Factor:Untreated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Untreated | Sample source:HY15549 Factor:Untreated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Factor:Treated | Sample source:HY15549 Meaib_ 174 5034214 5518265 456331 698063 1405428 2279737 3387421 4640581 5736322 6613280 6918267 7723013 6608788 6159125 403740 712668 1274789 1434548 3374428 3628176 5092717 6123275 5204194 6706309 5971197 6026616 536685 862207 1372858 1798506 3110878 3724419 5268758 5577380 6684029 6414067 Ala_ 232 16022720 12879114 73487911 70806436 62856354 56249707 44108422 40725112 31509571 24538593 19694746 21149027 24298245 22758021 77996756 74035088 70649639 52596906 57092539 46110011 39486171 37593467 24610620 30534295 16787296 15889651 82645204 78786633 68657917 61690815 49314703 37621561 34311954 28792690 22593013 17924309 Ala_ 260 13659186 11345012 63904735 61768860 54605748 49090415 38806605 35965396 27959323 21713595 17428954 18739744 21573651 20103543 67707960 64743153 61310825 46006889 49962777 40529224 34854359 33273036 21778757 27085097 14701861 14017799 72712524 68311210 60477929 53666314 44035563 33118850 30300364 25421616 19912131 15754011 13CAla_263 2346648.155 2964451.636 1399513.697 1568929.044 1698238.763 1850708.646 1948091.571 2600298.131 2826687.555 2646887.231 3065753.009 3122041.35 3287824.412 3192442.628 1523429.1 1650950.402 1845455.833 1693053.515 2278302.631 2415541.75 2589678.874 3097719.652 2349927.88 3209583.995 3743093.811 3542297.807 1970509.4 2158634.236 2370734.817 2602816.229 2721397.793 3417865.32 3563322.806 3653086.219 3649893.612 3563557.288 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z rt (min) Meaib_ 174 174 13.71 Ala_ 232 232 18.25 Ala_ 260 260 18.25 13CAla_263 263 18.25 METABOLITES_END #END