Summary of Study ST003429

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 PR001941. The data can be accessed directly via it's Project DOI: 10.21228/M8TM76 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.

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Study IDST003429
Study TitleIntracellular and medium metabolomics of BT-474 cells treated with Fasnall that was manufactured by Enamine
Study SummaryBT-474 human breast cancer cells were treated with various concentrations of Fasnall for 24 h. The drug was manufactured by Enamine (1-benzyl-N-{5,6-dimethylthieno[2,3-d]pyrimidin-4-yl}pyrrolidin-3-amine, EN300-14327544).
Institute
Wistar Institute
DepartmentMolecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center
LaboratorySchug's Lab
Last NameMukha
First NameDzmitry
Address3601 Spruce St, Philadelphia, PA 19104, USA
Emaildmukha@wistar.org
Phone+12154956903
Submit Date2024-08-21
Num Groups15
Total Subjects45
PublicationsSubmission Pending
Raw Data AvailableYes
Raw Data File Type(s)mzML, wiff
Analysis Type DetailLC-MS
Release Date2024-09-12
Release Version1
Dzmitry Mukha Dzmitry Mukha
https://dx.doi.org/10.21228/M8TM76
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001941
Project DOI:doi: 10.21228/M8TM76
Project Title:The shutdown of NADH oxidation via Respiratory Complex I mimics fatty acid biosynthesis inhibition
Project Type:LC-MS Quantitative Analysis
Project Summary:Proliferating cancer cells actively utilize anabolic processes for biomass production, including de novo biosynthesis of amino acids, nucleotides, and fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid synthase (FASN), is widely recognized as a promising therapeutic target in cancer and other health conditions. Here, we establish a metabolic signature of FASN inhibition using a panel of pharmacological inhibitors (GSK2194069, TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of some commonly used FASN inhibitors is inconsistent with the metabolic signature of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A, succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I inhibitor that mimics FASN inhibition through NADH accumulation and consequent depletion of the tricarboxylic acid cycle metabolites. We demonstrate that Fasnall impairs tumor growth in several oxidative phosphorylation-dependent cancer models, including combination therapy-resistant melanoma patient-derived xenografts. Fasnall administration does not reproduce neurological side effects in mice reported for other Complex I inhibitors. Our results have significant implications for understanding the FASN role in human health and disease and provide evidence of therapeutic potential for Complex I inhibitors with fast systemic clearance. Our findings also highlight the continuing need for validation of small molecule inhibitors to distinguish high-quality chemical probes and to expand the understanding of their application.
Institute:Wistar Institute
Department:Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer Center
Laboratory:Schug's Lab
Last Name:Mukha
First Name:Dzmitry
Address:3601 Spruce St., Philadelphia, Pennsylvania 19104, USA
Email:dmukha@wistar.org
Phone:+12154956903
Funding Source:This work was supported by grants from the National Institutes of Health (NIH) National Cancer Institute (NCI) DP2 CA249950-01 (Z.T.S.), NIH NCI P01 CA114046 (Z.T.S.), Melanoma Research Foundation 717173 (Z.T.S.), and Susan G. Komen CCR19608782 (Z.T.S.).
Publications:Submission Pending
Contributors:Dzmitry Mukha, Jena Dessain, Seamus O’Connor, Katherine Pniewski, Fabrizio Bertolazzi, Jeet Patel, Mary Mullins, Zachary T. Schug

Subject:

Subject ID:SU003556
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:60
Gender:Female
Cell Strain Details:BT-474, breast cancer cell line
Species Group:Mammals

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Sample Type Drug Treatment
SA37782211 BT-474 Fasnall 0.1 uM cellsBT-474 breast cancer cells Intracellular 0.1 uM Fasnall
SA37782304 BT-474 Fasnall 0.1 uM cellsBT-474 breast cancer cells Intracellular 0.1 uM Fasnall
SA37782418 BT-474 Fasnall 0.1 uM cellsBT-474 breast cancer cells Intracellular 0.1 uM Fasnall
SA37782821 BT-474 Fasnall 10 uM cellsBT-474 breast cancer cells Intracellular 10 uM Fasnall
SA37782914 BT-474 Fasnall 10 uM cellsBT-474 breast cancer cells Intracellular 10 uM Fasnall
SA37783007 BT-474 Fasnall 10 uM cellsBT-474 breast cancer cells Intracellular 10 uM Fasnall
SA37782505 BT-474 Fasnall 1 uM cellsBT-474 breast cancer cells Intracellular 1 uM Fasnall
SA37782619 BT-474 Fasnall 1 uM cellsBT-474 breast cancer cells Intracellular 1 uM Fasnall
SA37782712 BT-474 Fasnall 1 uM cellsBT-474 breast cancer cells Intracellular 1 uM Fasnall
SA37783108 BT-474 Fasnall 20 uM cellsBT-474 breast cancer cells Intracellular 20 uM Fasnall
SA37783215 BT-474 Fasnall 20 uM cellsBT-474 breast cancer cells Intracellular 20 uM Fasnall
SA37783322 BT-474 Fasnall 20 uM cellsBT-474 breast cancer cells Intracellular 20 uM Fasnall
SA37783423 BT-474 Fasnall 40 uM cellsBT-474 breast cancer cells Intracellular 40 uM Fasnall
SA37783516 BT-474 Fasnall 40 uM cellsBT-474 breast cancer cells Intracellular 40 uM Fasnall
SA37783609 BT-474 Fasnall 40 uM cellsBT-474 breast cancer cells Intracellular 40 uM Fasnall
SA37783706 BT-474 Fasnall 5 uM cellsBT-474 breast cancer cells Intracellular 5 uM Fasnall
SA37783820 BT-474 Fasnall 5 uM cellsBT-474 breast cancer cells Intracellular 5 uM Fasnall
SA37783913 BT-474 Fasnall 5 uM cellsBT-474 breast cancer cells Intracellular 5 uM Fasnall
SA37784010 BT-474 Fasnall 0 uM cellsBT-474 breast cancer cells Intracellular Control
SA37784103 BT-474 Fasnall 0 uM cellsBT-474 breast cancer cells Intracellular Control
SA37784217 BT-474 Fasnall 0 uM cellsBT-474 breast cancer cells Intracellular Control
SA37784333 BT-474 Fasnall 0.1 uM medBT-474 breast cancer cells Medium 0.1 uM Fasnall
SA37784425 BT-474 Fasnall 0.1 uM medBT-474 breast cancer cells Medium 0.1 uM Fasnall
SA37784541 BT-474 Fasnall 0.1 uM medBT-474 breast cancer cells Medium 0.1 uM Fasnall
SA37784929 BT-474 Fasnall 10 uM medBT-474 breast cancer cells Medium 10 uM Fasnall
SA37785045 BT-474 Fasnall 10 uM medBT-474 breast cancer cells Medium 10 uM Fasnall
SA37785137 BT-474 Fasnall 10 uM medBT-474 breast cancer cells Medium 10 uM Fasnall
SA37784642 BT-474 Fasnall 1 uM medBT-474 breast cancer cells Medium 1 uM Fasnall
SA37784734 BT-474 Fasnall 1 uM medBT-474 breast cancer cells Medium 1 uM Fasnall
SA37784826 BT-474 Fasnall 1 uM medBT-474 breast cancer cells Medium 1 uM Fasnall
SA37785246 BT-474 Fasnall 20 uM medBT-474 breast cancer cells Medium 20 uM Fasnall
SA37785338 BT-474 Fasnall 20 uM medBT-474 breast cancer cells Medium 20 uM Fasnall
SA37785430 BT-474 Fasnall 20 uM medBT-474 breast cancer cells Medium 20 uM Fasnall
SA37785531 BT-474 Fasnall 40 uM medBT-474 breast cancer cells Medium 40 uM Fasnall
SA37785647 BT-474 Fasnall 40 uM medBT-474 breast cancer cells Medium 40 uM Fasnall
SA37785739 BT-474 Fasnall 40 uM medBT-474 breast cancer cells Medium 40 uM Fasnall
SA37785843 BT-474 Fasnall 5 uM medBT-474 breast cancer cells Medium 5 uM Fasnall
SA37785927 BT-474 Fasnall 5 uM medBT-474 breast cancer cells Medium 5 uM Fasnall
SA37786035 BT-474 Fasnall 5 uM medBT-474 breast cancer cells Medium 5 uM Fasnall
SA37786140 BT-474 Fasnall 0 uM medBT-474 breast cancer cells Medium Control
SA37786224 BT-474 Fasnall 0 uM medBT-474 breast cancer cells Medium Control
SA37786332 BT-474 Fasnall 0 uM medBT-474 breast cancer cells Medium Control
SA37786449 BlankNA Blank NA
SA37786501 BlankNA Blank NA
SA37786644 Bottle mediumNA Medium 10 uM Fasnall
SA37786736 Bottle mediumNA Medium 10 uM Fasnall
SA37786828 Bottle mediumNA Medium 10 uM Fasnall
SA37786902 DMEM/F12 no addsNA QC NA
SA37787048 DMEM/F12 no addsNA QC NA
Showing results 1 to 49 of 49

Collection:

Collection ID:CO003549
Collection Summary:For intracellular metabolite samples, the medium was aspirated, and cells were washed with PBS volume matching the volume of the medium. Metabolites were extracted with ice-cold 80% methanol. The volume of the solvent was 500 µl per 6-cm Petri dish (scaled according to the ratio of surface areas for other cell containers). After adding the methanol solution, cells were scraped from the plates, and all the content was transferred to Eppendorf tubes.
Collection Protocol Filename:DM_metabolomics_samples.txt
Sample Type:Breast cancer cells
Collection Method:80% methanol extraction
Storage Conditions:-80℃
Collection Vials:1.5 ml plastic centrifuge tubes
Storage Vials:1.5 ml plastic centrifuge tubes

Treatment:

Treatment ID:TR003565
Treatment Summary:Cells were grown in RPMI-1640 supplemented with 10% dialyzed FBS and treated with various concentrations of rotenone for 24 h.
Treatment Compound:Fasnall (1-benzyl-N-{5,6-dimethylthieno[2,3-d]pyrimidin-4-yl}pyrrolidin-3-amine, Enamine EN300-14327544)
Treatment Vehicle:DMSO
Cell Growth Container:6-cm Petri dishes
Cell Media:RPMI-1640
Cell Envir Cond:37C, 5% CO2
Cell Pct Confluence:~70%
Cell Media Lastchanged:24 h before collection

Sample Preparation:

Sampleprep ID:SP003563
Sampleprep Summary:Intracellular metabolites were extracted with ice-cold 80% methanol, and medium samples were extracted with 100% methanol. The samples were centrifuged at 18,000 g 4C for 20 min. After transferring the supernatant to new tubes, centrifugation was repeated with the same parameters.
Sampleprep Protocol Filename:DM_metabolomics_samples.txt
Processing Storage Conditions:4℃
Extraction Method:80% methanol
Extract Enrichment:None
Extract Cleanup:None
Extract Storage:-80℃
Sample Resuspension:None
Sample Derivatization:None
Sample Spiking:None
Subcellular Location:Intracellular metabolites and medium metabolites

Combined analysis:

Analysis ID AN005631 AN005632
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Shimadzu 20AD Shimadzu 20AD
Column Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap ABI Sciex 5500 QTrap
Ion Mode NEGATIVE POSITIVE
Units Counts per second (cps) Counts per second (cps)

Chromatography:

Chromatography ID:CH004278
Instrument Name:Shimadzu 20AD
Column Name:Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Pressure:900-3000 psi
Column Temperature:40
Flow Gradient:0-12.5 min, 80-30% B; 12.5-15 min, 30% B; 15-15.2 min, 30-80% B; 15.2-22.5 min, 80% B
Flow Rate:0-20 min, 0.2 ml/min; 20-21 min 0.2-0.3 ml/min; 21-22 min, 0.3 ml/min; 22-22.1 min, 0.2 ml/min; 22.1-22.5 min, 0.2 ml/min
Injection Temperature:4
Sample Injection:1-5 ul
Solvent A:100% Water; 0.01% ammonium hydroxide; 20 mM ammonium bicarbonate
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS005355
Analysis ID:AN005631
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Data were analyzed with SCIEX Multiquant 3.0.3.
Ion Mode:NEGATIVE
Capillary Temperature:500 °C
Capillary Voltage:-4500
Dry Gas Flow:70
Dry Gas Temp:500 °C
Ion Source Temperature:500 °C
Ion Spray Voltage:-4500
Mass Accuracy:UNIT
Source Temperature:500 °C
Spray Voltage:-4500
Desolvation Gas Flow:70
Desolvation Temperature:500 °C
  
MS ID:MS005356
Analysis ID:AN005632
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Data were analyzed with SCIEX Multiquant 3.0.3.
Ion Mode:POSITIVE
Capillary Temperature:500 °C
Capillary Voltage:4500
Dry Gas Flow:70
Dry Gas Temp:500 °C
Ion Source Temperature:500 °C
Ion Spray Voltage:4500
Mass Accuracy:UNIT
Source Temperature:500 °C
Spray Voltage:4500
Desolvation Gas Flow:70
Desolvation Temperature:500 °C
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