Summary of Study ST003435

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 IDST003435
Study TitleMetabolomics analysis of zebrafish embryos treated with rotenone, Fasnall, TVB-2640, and GSK2194069
Study SummaryTo compare the metabolic effects and toxicity of Fasnall with rotenone in vivo, zebrafish embryos 48 h post-fertilization were exposed to a drug-containing medium for 6 h. Rotenone at 25 nM is lethal to fish embryos, while 5 nM concentration leads to a ~15-fold lactate accumulation. Similarly, Fasnall treatment increases lactate content, although the magnitude of the effect is significantly lower. Unlike 5 nM rotenone, Fasnall treatment does not cause visible phenotypic changes in the yolk. The zebrafish model suggests that Fasnall acts as a Complex I inhibitor in vivo.
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 Groups16
Total Subjects48
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

Select appropriate tab below to view additional metadata details:

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:SU003562
Subject Type:Fish
Subject Species:Danio rerio
Taxonomy ID:7955
Gender:Pooled

Factors:

Subject type: Fish; Subject species: Danio rerio (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Sample Type Drug Treatment
SA37812524 BlankNA Blank NA
SA37812649 BlankNA Blank NA
SA37812701 BlankNA Blank NA
SA37812802 DMEM/F12 no addsNA QC NA
SA37812948 DMEM/F12 no addsNA QC NA
SA37813036 13-1 Rotenone 1 nM Medium 1Zebrafish medium Zebrafish medium 1 nM rotenone
SA37813138 13-3 Rotenone 1 nM Medium 3Zebrafish medium Zebrafish medium 1 nM rotenone
SA37813237 13-2 Rotenone 1 nM Medium 2Zebrafish medium Zebrafish medium 1 nM rotenone
SA37813343 21-2 Fasnall 20 uM Medium 2Zebrafish medium Zebrafish medium 20 uM Fasnall
SA37813444 21-3 Fasnall 20 uM Medium 3Zebrafish medium Zebrafish medium 20 uM Fasnall
SA37813542 21-1 Fasnall 20 uM Medium 1Zebrafish medium Zebrafish medium 20 uM Fasnall
SA37813645 25-1 Fasnall 40 uM Medium 1Zebrafish medium Zebrafish medium 40 uM Fasnall
SA37813746 25-2 Fasnall 40 uM Medium 2Zebrafish medium Zebrafish medium 40 uM Fasnall
SA37813847 25-3 Fasnall 40 uM Medium 3Zebrafish medium Zebrafish medium 40 uM Fasnall
SA37813931 05-2 GSK 40 uM Medium 2Zebrafish medium Zebrafish medium 40 uM GSK2194069
SA37814030 05-1 GSK 40 uM Medium 1Zebrafish medium Zebrafish medium 40 uM GSK2194069
SA37814132 05-3 GSK 40 uM Medium 3Zebrafish medium Zebrafish medium 40 uM GSK2194069
SA37814234 09-2 TVB-2640 40 uM Medium 2Zebrafish medium Zebrafish medium 40 uM TVB-2640
SA37814333 09-1 TVB-2640 40 uM Medium 1Zebrafish medium Zebrafish medium 40 uM TVB-2640
SA37814435 09-3 TVB-2640 40 uM Medium 3Zebrafish medium Zebrafish medium 40 uM TVB-2640
SA37814540 17-2 Rotenone 5 nM Medium 2Zebrafish medium Zebrafish medium 5 nM rotenone
SA37814641 17-3 Rotenone 5 nM Medium 3Zebrafish medium Zebrafish medium 5 nM rotenone
SA37814739 17-1 Rotenone 5 nM Medium 1Zebrafish medium Zebrafish medium 5 nM rotenone
SA37814829 01-3 DMSO Medium 3Zebrafish medium Zebrafish medium Vehicle
SA37814928 01-2 DMSO Medium 2Zebrafish medium Zebrafish medium Vehicle
SA37815027 01-1 DMSO Medium 1Zebrafish medium Zebrafish medium Vehicle
SA37815106 14 Rotenone 1 nM 1Zebrafish Zebrafish embryo metabolite extract 1 nM rotenone
SA37815220 16 Rotenone 1 nM 3 *Zebrafish Zebrafish embryo metabolite extract 1 nM rotenone
SA37815313 15 Rotenone 1 nM 2Zebrafish Zebrafish embryo metabolite extract 1 nM rotenone
SA37815422 24 Fasnall 20 uM 3Zebrafish Zebrafish embryo metabolite extract 20 uM Fasnall
SA37815515 23 Fasnall 20 uM 2Zebrafish Zebrafish embryo metabolite extract 20 uM Fasnall
SA37815608 22 Fasnall 20 uM 1Zebrafish Zebrafish embryo metabolite extract 20 uM Fasnall
SA37815716 27 Fasnall 40 uM 2Zebrafish Zebrafish embryo metabolite extract 40 uM Fasnall
SA37815809 26 Fasnall 40 uM 1Zebrafish Zebrafish embryo metabolite extract 40 uM Fasnall
SA37815923 28 Fasnall 40 uM 3Zebrafish Zebrafish embryo metabolite extract 40 uM Fasnall
SA37816025 08 GSK 40 uM 3Zebrafish Zebrafish embryo metabolite extract 40 uM GSK2194069
SA37816104 06 GSK 40 uM 1Zebrafish Zebrafish embryo metabolite extract 40 uM GSK2194069
SA37816218 07 GSK 40 uM 2Zebrafish Zebrafish embryo metabolite extract 40 uM GSK2194069
SA37816311 07 GSK 40 uM 2Zebrafish Zebrafish embryo metabolite extract 40 uM GSK2194069
SA37816405 10 TVB-2640 40 uM 1Zebrafish Zebrafish embryo metabolite extract 40 uM TVB-2640
SA37816519 12 TVB-2640 40 uM 3Zebrafish Zebrafish embryo metabolite extract 40 uM TVB-2640
SA37816612 11 TVB-2640 40 uM 2Zebrafish Zebrafish embryo metabolite extract 40 uM TVB-2640
SA37816721 19 Rotenone 5 nM 2Zebrafish Zebrafish embryo metabolite extract 5 nM rotenone
SA37816814 19 Rotenone 5 nM 2Zebrafish Zebrafish embryo metabolite extract 5 nM rotenone
SA37816907 18 Rotenone 5 nM 1Zebrafish Zebrafish embryo metabolite extract 5 nM rotenone
SA37817026 20 Rotenone 5 nM 3Zebrafish Zebrafish embryo metabolite extract 5 nM rotenone
SA37817117 04 DMSO 3Zebrafish Zebrafish embryo metabolite extract Vehicle
SA37817210 03 DMSO 2Zebrafish Zebrafish embryo metabolite extract Vehicle
SA37817303 02 DMSO 1Zebrafish Zebrafish embryo metabolite extract Vehicle
Showing results 1 to 49 of 49

Collection:

Collection ID:CO003555
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:Media, Metabolite extract
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:TR003571
Treatment Summary:The zebrafish research was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Husbandry was performed in accordance with institutional animal welfare guidelines. Embryos from wild type, Tübingen zebrafish were collected within 15 minutes of fertilization. Embryos between 2 and 3 crosses were equally pooled and allocated to treatment conditions for all experiments. Embryos were reared at 28 °C in E3 medium (4 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, no methylene blue) until treatment. Pharmacological agents were diluted from stocks at room temperature in E3 for 15 minutes before treatment. For treatment at 48 h post-fertilization, embryos in their chorions were transferred to clean plates with the inhibitor-supplemented E3 and incubated for 6 hours.
Treatment Compound:Rotenone, Fasnall, TVB-2640, and GSK2194069
Treatment Route:Drugs were dissolved in the medium
Treatment Vehicle:DMSO

Sample Preparation:

Sampleprep ID:SP003569
Sampleprep Summary:Zebrafish embryos were combined by eight per Eppendorf tube, washed with PBS twice, and snap-frozen on dry ice. Frozen samples were ground at the temperature of liquid nitrogen by Retsch Cryomill. To each tube, 300 µl of 80% methanol were added.
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 AN005643 AN005644
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:CH004284
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:MS005367
Analysis ID:AN005643
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:MS005368
Analysis ID:AN005644
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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