Summary of Study ST003713
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 PR002306. The data can be accessed directly via it's Project DOI: 10.21228/M8JR73 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST003713 |
| Study Title | Photoaffinity ligand of Cystic Fibrosis corrector VX-445 identifies SCCPDH as an off-target |
| Study Type | untargeted metabolomics analysis |
| Study Summary | We utilized LC-MS/MS to identify crosslinked proteins in CF bronchial epithelial cells expressing F508del CFTR, identifying SCCPDH, an uncharacterized putative oxidoreductase, as a VX-445 specific off-target. We then characterized changes in the metabolomic profiles of cells overexpressing SCCPDH to decipher its role and implications of VX-445 binding to SCCPDH-dependent metabolic changes. We found dysregulation of amino acid metabolism and a potential inhibitory activity of VX-445 on SCCPDH. The identified off-target may explain exacerbation of psychological symptoms observed in patients on HEMT, thus emphasizing the need for further optimization of corrector combinations. |
| Institute | Vanderbilt University |
| Department | Chemistry |
| Laboratory | Center for Innovative Technology |
| Last Name | CODREANU |
| First Name | GABRIELA SIMONA |
| Address | 1234 STEVENSON CENTER LANE |
| SIMONA.CODREANU@VANDERBILT.EDU | |
| Phone | 6158758422 |
| Submit Date | 2025-02-07 |
| Num Groups | 3 |
| Total Subjects | 15 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-08-07 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002306 |
| Project DOI: | doi: 10.21228/M8JR73 |
| Project Title: | Photoaffinity ligand of Cystic Fibrosis corrector VX-445 identifies SCCPDH as an off-target |
| Project Type: | Untargeted Metabolomics analysis |
| Project Summary: | Cystic fibrosis (CF) pharmacological correctors such as VX-445 are used in combinations as highly effective modulator therapy (HEMT) at CF clinics due to their efficacy in improving Cystic Fibrosis transmembrane conductance regulator (CFTR) protein trafficking and function. We utilized LC-MS/MS to identify crosslinked proteins in CF bronchial epithelial cells expressing F508del CFTR, identifying SCCPDH, an uncharacterized putative oxidoreductase, as a VX-445 specific off-target. We then characterized changes in the metabolomic profiles of cells overexpressing SCCPDH to decipher its role and implications of VX-445 binding to SCCPDH-dependent metabolic changes. We found dysregulation of amino acid metabolism and a potential inhibitory activity of VX-445 on SCCPDH. The identified off-target may explain exacerbation of psychological symptoms observed in patients on HEMT, thus emphasizing the need for further optimization of corrector combinations. |
| Institute: | Vanderbilt University |
| Department: | Chemistry |
| Laboratory: | Center for Innovative Technology |
| Last Name: | CODREANU |
| First Name: | GABRIELA SIMONA |
| Address: | 1234 STEVENSON CENTER LANE |
| Email: | SIMONA.CODREANU@VANDERBILT.EDU |
| Phone: | 6158758422 |
Subject:
| Subject ID: | SU003845 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | HEK293T cells |
| Cell Strain Details: | HEK293T cells transfected with mock or SCCPDH plasmid and seeded in 6-well plates to be harvested at confluency. |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA406614 | T2_01 | SCCPDH-Myc_DMSO |
| SA406615 | T2_02 | SCCPDH-Myc_DMSO |
| SA406616 | T2_03 | SCCPDH-Myc_DMSO |
| SA406617 | T2_04 | SCCPDH-Myc_DMSO |
| SA406618 | T2_05 | SCCPDH-Myc_DMSO |
| SA406619 | CTR_01 | WT_GFP_DMSO |
| SA406620 | CTR_02 | WT_GFP_DMSO |
| SA406621 | CTR_03 | WT_GFP_DMSO |
| SA406622 | CTR_04 | WT_GFP_DMSO |
| SA406623 | CTR_05 | WT_GFP_DMSO |
| SA406624 | T1_01 | WT_GFP_VX-445 |
| SA406625 | T1_02 | WT_GFP_VX-446 |
| SA406626 | T1_03 | WT_GFP_VX-447 |
| SA406627 | T1_04 | WT_GFP_VX-448 |
| SA406628 | T1_05 | WT_GFP_VX-449 |
| Showing results 1 to 15 of 15 |
Collection:
| Collection ID: | CO003838 |
| Collection Summary: | Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s modification of Eagle’s medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% L-glutamine (200 mM, Gibco), and 1% penicillin/streptomycin (10,000 U; 10,000 μg/mL, Gibco). Cells were maintained in a 37°C humidified incubator at 5% CO2 – 95% air. Plasmids used for transient transfection expressed WT or SCCPDH-Myc (Sino Biological, HG17073-CM) in the pCMV3 vector, and the treatment was done with the second-generation corrector VX-445 (Elexacaftor) that can treat a wide range of Cystic fibrosis (CF)-causing variants. HEK293T cells transiently transfected with GFP, were either treated with DMSO or VX-445 (3 µM) or cells were transfected with saccharopine dehydrogenase-like oxidoreductase Myc tagged (SCCPDH-Myc) and treated with DMSO for 24 h (n = 5). Cells were scraped with ice cold ammonium formate (50 mM) and centrifuged at 200×g for 3 min at 4°C. Cell pellets were frozen at -80°C until further processing. |
| Sample Type: | HEK cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003854 |
| Treatment Summary: | HEK293T cells transiently transfected with GFP, were either treated with DMSO or VX-445 (3 µM) or cells were transfected with saccharopine dehydrogenase-like oxidoreductase Myc tagged (SCCPDH-Myc) and treated with DMSO for 24 h (n = 5). Cells were scraped with ice cold ammonium formate (50 mM) and centrifuged at 200×g for 3 min at 4°C. Cell pellets were frozen at -80°C until further processing. |
| Treatment Compound: | corrector VX-445 (Elexacaftor) |
| Treatment Dose: | 3uM |
Sample Preparation:
| Sampleprep ID: | SP003851 |
| Sampleprep Summary: | Samples were thawed on ice and lysed in 300 µL ice-cold lysis buffer (1:1:2, acetonitrile: methanol: ammonium bicarbonate 0.1M, pH 8.0) followed by probe tip sonication with 10 pulses at 30% power. Protein content was determined using a bicinchoninic acid protein assay (BCA assay, Thermo Fisher Scientific, Waltham, MA) and the appropriate amount of lysate was taken for 200µg total protein per sample and adjusted to 200 µL total volume with lysate buffer. Isotopically labeled standards (phenylalanine-D8 and biotin-D2) were added to each sample to determine later sample process variability as previously described. Following volume adjustment to 200 µL, 800 µL of cold MeOH was added to the samples. Individual samples were vortexed for 30 seconds and incubated overnight at -80°C for protein precipitation. Following incubation, samples were centrifuged for 10 min at 10,000 rpm at 4°C and the supernatant was transferred to a new labeled tube and dried down using a cold vacuum centrifuge. Samples were reconstituted in 100 µL H2O, 100 µL MeOH, and 10 µL of SPLASH LIPIDOMIX with vortex mixing after each addition. Samples were incubated at room temperature for 10 min followed by liquid-liquid extraction. For liquid-liquid extraction (LLE), 600 µL MTBE was added with vortex mixing for 30 seconds followed by incubation on ice for 10 min and centrifugation at 15,000 rpm for 15 minutes at 4°C. An upper (hydrophobic) fraction was transferred and dried down using cold vacuum centrifuge and stored at -80°C for further lipidomic studies. The lower (hydrophilic) fraction was transferred into a new Eppendorf tube, dried in vacuo, and stored at -80°C until further use. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Following standard addition, protein precipitation was performed by adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm for 10 min to eliminate proteins. The supernatants containing metabolites were dried via speed-vacuum. |
| Extract Cleanup: | LLE with MTBE |
| Extract Storage: | -80℃ |
Chromatography:
| Chromatography ID: | CH004625 |
| Chromatography Summary: | HILIC negative mode |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm) |
| Column Temperature: | 30℃ |
| 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 min 95%B |
| Flow Rate: | 0.20 mL/min |
| Solvent A: | 90% Water/10% Acetonitrile; 5mM Ammonium Formate; 0.1% Formic acid |
| Solvent B: | 90% Acetonitrile/10% Water; 5mM Ammonium Formate; 0.1% Formic acid |
| Chromatography Type: | HILIC |
Analysis:
| Analysis ID: | AN006094 |
| Analysis Type: | MS |
| Chromatography ID: | CH004625 |
| Has Mz: | 1 |
| Has Rt: | 1 |
| Rt Units: | Minutes |
| Results File: | ST003713_AN006094_Results.txt |
| Units: | peak intensity |
