#METABOLOMICS WORKBENCH Codreags00_20250207_124255 DATATRACK_ID:5604 STUDY_ID:ST003713 ANALYSIS_ID:AN006094 PROJECT_ID:PR002306 VERSION 1 CREATED_ON February 7, 2025, 3:51 pm #PROJECT PR:PROJECT_TITLE Photoaffinity ligand of Cystic Fibrosis corrector VX-445 identifies SCCPDH as an PR:PROJECT_TITLE off-target PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Cystic fibrosis (CF) pharmacological correctors such as VX-445 are used in PR:PROJECT_SUMMARY combinations as highly effective modulator therapy (HEMT) at CF clinics due to PR:PROJECT_SUMMARY their efficacy in improving Cystic Fibrosis transmembrane conductance regulator PR:PROJECT_SUMMARY (CFTR) protein trafficking and function. We utilized LC-MS/MS to identify PR:PROJECT_SUMMARY crosslinked proteins in CF bronchial epithelial cells expressing F508del CFTR, PR:PROJECT_SUMMARY identifying SCCPDH, an uncharacterized putative oxidoreductase, as a VX-445 PR:PROJECT_SUMMARY specific off-target. We then characterized changes in the metabolomic profiles PR:PROJECT_SUMMARY of cells overexpressing SCCPDH to decipher its role and implications of VX-445 PR:PROJECT_SUMMARY binding to SCCPDH-dependent metabolic changes. We found dysregulation of amino PR:PROJECT_SUMMARY acid metabolism and a potential inhibitory activity of VX-445 on SCCPDH. The PR:PROJECT_SUMMARY identified off-target may explain exacerbation of psychological symptoms PR:PROJECT_SUMMARY observed in patients on HEMT, thus emphasizing the need for further optimization PR:PROJECT_SUMMARY of corrector combinations. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME CODREANU PR:FIRST_NAME GABRIELA SIMONA PR:ADDRESS 1234 STEVENSON CENTER LANE PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Photoaffinity ligand of Cystic Fibrosis corrector VX-445 identifies SCCPDH as an ST:STUDY_TITLE off-target ST:STUDY_TYPE untargeted metabolomics analysis ST:STUDY_SUMMARY We utilized LC-MS/MS to identify crosslinked proteins in CF bronchial epithelial ST:STUDY_SUMMARY cells expressing F508del CFTR, identifying SCCPDH, an uncharacterized putative ST:STUDY_SUMMARY oxidoreductase, as a VX-445 specific off-target. We then characterized changes ST:STUDY_SUMMARY in the metabolomic profiles of cells overexpressing SCCPDH to decipher its role ST:STUDY_SUMMARY and implications of VX-445 binding to SCCPDH-dependent metabolic changes. We ST:STUDY_SUMMARY found dysregulation of amino acid metabolism and a potential inhibitory activity ST:STUDY_SUMMARY of VX-445 on SCCPDH. The identified off-target may explain exacerbation of ST:STUDY_SUMMARY psychological symptoms observed in patients on HEMT, thus emphasizing the need ST:STUDY_SUMMARY for further optimization of corrector combinations. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME CODREANU ST:FIRST_NAME GABRIELA SIMONA ST:ADDRESS 1234 STEVENSON CENTER LANE ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:PHONE 6158758422 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 15 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN HEK293T cells SU:CELL_STRAIN_DETAILS HEK293T cells transfected with mock or SCCPDH plasmid and seeded in 6-well SU:CELL_STRAIN_DETAILS plates to be harvested at confluency. #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 CTR_01 CTR_01 Sample source:HEK293T cells | Genotype:WT_GFP_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_CTR_01 SUBJECT_SAMPLE_FACTORS CTR_02 CTR_02 Sample source:HEK293T cells | Genotype:WT_GFP_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_CTR_02 SUBJECT_SAMPLE_FACTORS CTR_03 CTR_03 Sample source:HEK293T cells | Genotype:WT_GFP_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_CTR_03 SUBJECT_SAMPLE_FACTORS CTR_04 CTR_04 Sample source:HEK293T cells | Genotype:WT_GFP_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_CTR_04 SUBJECT_SAMPLE_FACTORS CTR_05 CTR_05 Sample source:HEK293T cells | Genotype:WT_GFP_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_CTR_05 SUBJECT_SAMPLE_FACTORS T1_01 T1_01 Sample source:HEK293T cells | Genotype:WT_GFP_VX-445 RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T1_01 SUBJECT_SAMPLE_FACTORS T1_02 T1_02 Sample source:HEK293T cells | Genotype:WT_GFP_VX-446 RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T1_02 SUBJECT_SAMPLE_FACTORS T1_03 T1_03 Sample source:HEK293T cells | Genotype:WT_GFP_VX-447 RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T1_03 SUBJECT_SAMPLE_FACTORS T1_04 T1_04 Sample source:HEK293T cells | Genotype:WT_GFP_VX-448 RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T1_04 SUBJECT_SAMPLE_FACTORS T1_05 T1_05 Sample source:HEK293T cells | Genotype:WT_GFP_VX-449 RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T1_05 SUBJECT_SAMPLE_FACTORS T2_01 T2_01 Sample source:HEK293T cells | Genotype:SCCPDH-Myc_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T2_01 SUBJECT_SAMPLE_FACTORS T2_02 T2_02 Sample source:HEK293T cells | Genotype:SCCPDH-Myc_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T2_02 SUBJECT_SAMPLE_FACTORS T2_03 T2_03 Sample source:HEK293T cells | Genotype:SCCPDH-Myc_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T2_03 SUBJECT_SAMPLE_FACTORS T2_04 T2_04 Sample source:HEK293T cells | Genotype:SCCPDH-Myc_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T2_04 SUBJECT_SAMPLE_FACTORS T2_05 T2_05 Sample source:HEK293T cells | Genotype:SCCPDH-Myc_DMSO RAW_FILE_NAME(MS Sample Name)=SC_20241030_aHILICn_FMS_Plate_T2_05 #COLLECTION CO:COLLECTION_SUMMARY Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s CO:COLLECTION_SUMMARY modification of Eagle’s medium (DMEM, Corning) supplemented with 10% fetal CO:COLLECTION_SUMMARY bovine serum (FBS, Gibco), 1% L-glutamine (200 mM, Gibco), and 1% CO:COLLECTION_SUMMARY penicillin/streptomycin (10,000 U; 10,000 μg/mL, Gibco). Cells were maintained CO:COLLECTION_SUMMARY in a 37 °C humidified incubator at 5% CO2 – 95% air. Plasmids used for CO:COLLECTION_SUMMARY transient transfection expressed WT or SCCPDH-Myc (Sino Biological, HG17073-CM) CO:COLLECTION_SUMMARY in the pCMV3 vector, and the treatment was done with the second-generation CO:COLLECTION_SUMMARY corrector VX-445 (Elexacaftor) that can treat a wide range of Cystic fibrosis CO:COLLECTION_SUMMARY (CF)-causing variants. HEK293T cells transiently transfected with GFP, were CO:COLLECTION_SUMMARY either treated with DMSO or VX-445 (3 µM) or cells were transfected with CO:COLLECTION_SUMMARY saccharopine dehydrogenase-like oxidoreductase Myc tagged (SCCPDH-Myc) and CO:COLLECTION_SUMMARY treated with DMSO for 24 h (n = 5). Cells were scraped with ice cold ammonium CO:COLLECTION_SUMMARY formate (50 mM) and centrifuged at 200×g for 3 min at 4 °C. Cell pellets were CO:COLLECTION_SUMMARY frozen at -80 °C until further processing. CO:SAMPLE_TYPE HEK cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY HEK293T cells transiently transfected with GFP, were either treated with DMSO or TR:TREATMENT_SUMMARY VX-445 (3 µM) or cells were transfected with saccharopine dehydrogenase-like TR:TREATMENT_SUMMARY oxidoreductase Myc tagged (SCCPDH-Myc) and treated with DMSO for 24 h (n = 5). TR:TREATMENT_SUMMARY Cells were scraped with ice cold ammonium formate (50 mM) and centrifuged at TR:TREATMENT_SUMMARY 200×g for 3 min at 4 °C. Cell pellets were frozen at -80 °C until further TR:TREATMENT_SUMMARY processing. TR:TREATMENT_COMPOUND corrector VX-445 (Elexacaftor) TR:TREATMENT_DOSE 3uM #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were thawed on ice and lysed in 300 µL ice-cold lysis buffer (1:1:2, SP:SAMPLEPREP_SUMMARY acetonitrile: methanol: ammonium bicarbonate 0.1M, pH 8.0) followed by probe tip SP:SAMPLEPREP_SUMMARY sonication with 10 pulses at 30% power. Protein content was determined using a SP:SAMPLEPREP_SUMMARY bicinchoninic acid protein assay (BCA assay, Thermo Fisher Scientific, Waltham, SP:SAMPLEPREP_SUMMARY MA) and the appropriate amount of lysate was taken for 200µg total protein per SP:SAMPLEPREP_SUMMARY sample and adjusted to 200 µL total volume with lysate buffer. Isotopically SP:SAMPLEPREP_SUMMARY labeled standards (phenylalanine-D8 and biotin-D2) were added to each sample to SP:SAMPLEPREP_SUMMARY determine later sample process variability as previously described. Following SP:SAMPLEPREP_SUMMARY volume adjustment to 200 µL, 800 µL of cold MeOH was added to the samples. SP:SAMPLEPREP_SUMMARY Individual samples were vortexed for 30 seconds and incubated overnight at -80 SP:SAMPLEPREP_SUMMARY °C for protein precipitation. Following incubation, samples were centrifuged SP:SAMPLEPREP_SUMMARY for 10 min at 10,000 rpm at 4 °C and the supernatant was transferred to a new SP:SAMPLEPREP_SUMMARY labeled tube and dried down using a cold vacuum centrifuge. Samples were SP:SAMPLEPREP_SUMMARY reconstituted in 100 µL H2O, 100 µL MeOH, and 10 µL of SPLASH LIPIDOMIX with SP:SAMPLEPREP_SUMMARY vortex mixing after each addition. Samples were incubated at room temperature SP:SAMPLEPREP_SUMMARY for 10 min followed by liquid-liquid extraction. For liquid-liquid extraction SP:SAMPLEPREP_SUMMARY (LLE), 600 µL MTBE was added with vortex mixing for 30 seconds followed by SP:SAMPLEPREP_SUMMARY incubation on ice for 10 min and centrifugation at 15,000 rpm for 15 minutes at SP:SAMPLEPREP_SUMMARY 4 °C. An upper (hydrophobic) fraction was transferred and dried down using cold SP:SAMPLEPREP_SUMMARY vacuum centrifuge and stored at -80 °C for further lipidomic studies. The lower SP:SAMPLEPREP_SUMMARY (hydrophilic) fraction was transferred into a new Eppendorf tube, dried in SP:SAMPLEPREP_SUMMARY vacuo, and stored at -80 °C until further use. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following standard addition, protein precipitation was performed by adding SP:EXTRACTION_METHOD 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C SP:EXTRACTION_METHOD overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 SP:EXTRACTION_METHOD min to eliminate proteins. The supernatants containing metabolites were dried SP:EXTRACTION_METHOD via speed-vacuum. SP:EXTRACT_CLEANUP LLE with MTBE SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC negative mode CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1mm,2.5um) CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:FLOW_GRADIENT 30 min; 0-1 min 95%B, 1-12 min 95-45%B, 12-15 min 45%B, 15-26 min 45-95%B, 26-30 CH:FLOW_GRADIENT min 95%B CH:FLOW_RATE 0.20mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK) was used to review, MS:MS_COMMENTS process and normalize the mass spectrometry data. The pooled QC sample was used MS:MS_COMMENTS to align all MS and MS/MS sample runs. MS:MS_RESULTS_FILE ST003713_AN006094_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END