Summary of Study ST003412

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.

Study ID	ST003412
Study Title	Intracellular and medium metabolomics for BT-474 cells treated with cerulenin, TVB-2640, and TVB-3166 for 24 h
Study Type	Intracellular metabolomics, medium metabolomics
Study Summary	Intracellular and medium metabolomics for BT-474 cells treated with cerulenin, TVB-2640, and TVB-3166 for 24 h
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, PA 19104, USA
Email	dmukha@wistar.org
Phone	+12154956903
Submit Date	2024-08-09
Num Groups	45
Total Subjects	135
Publications	Submission Pending
Raw Data Available	Yes
Raw Data File Type(s)	mzML, wiff
Analysis Type Detail	LC-MS
Release Date	2024-08-26
Release Version	1

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:	SU003538
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

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	Drug Treatment
SA376311	29 BT474 Cerulenin 0.5 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	0.5 uM cerulenin
SA376312	36 BT474 Cerulenin 0.5 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	0.5 uM cerulenin
SA376313	43 BT474 Cerulenin 0.5 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	0.5 uM cerulenin
SA376314	30 BT474 Cerulenin 2.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	2.0 uM cerulenin
SA376315	44 BT474 Cerulenin 2.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	2.0 uM cerulenin
SA376316	37 BT474 Cerulenin 2.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	2.0 uM cerulenin
SA376317	40 BT474 Cerulenin 20 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	20 uM cerulenin
SA376318	33 BT474 Cerulenin 20 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	20 uM cerulenin
SA376319	47 BT474 Cerulenin 20 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	20 uM cerulenin
SA376320	38 BT474 Cerulenin 4.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	4.0 uM cerulenin
SA376321	31 BT474 Cerulenin 4.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	4.0 uM cerulenin
SA376322	45 BT474 Cerulenin 4.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	4.0 uM cerulenin
SA376323	48 BT474 Cerulenin 50 uM cells (dead)	BT-474 Breast cancer cells	Intracellular	cerulenin	50 uM cerulenin
SA376324	34 BT474 Cerulenin 50 uM cells (dead)	BT-474 Breast cancer cells	Intracellular	cerulenin	50 uM cerulenin
SA376325	41 BT474 Cerulenin 50 uM cells (dead)	BT-474 Breast cancer cells	Intracellular	cerulenin	50 uM cerulenin
SA376326	46 BT474 Cerulenin 6.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	6.0 uM cerulenin
SA376327	32 BT474 Cerulenin 6.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	6.0 uM cerulenin
SA376328	39 BT474 Cerulenin 6.0 uM cells	BT-474 Breast cancer cells	Intracellular	cerulenin	6.0 uM cerulenin
SA376266	131 BT474 TVB-3166 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376267	76 BT474 TVB-2640 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376268	90 BT474 TVB-2640 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376269	83 BT474 TVB-2640 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376270	35 BT474 Cerulenin 0 uM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376271	28 BT474 Cerulenin 0 uM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376272	124 BT474 TVB-3166 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376273	42 BT474 Cerulenin 0 uM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376274	138 BT474 TVB-3166 0 nM cells	BT-474 Breast cancer cells	Intracellular	NA	Control
SA376275	77 BT474 TVB-2640 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	1 nM TVB-2640
SA376276	84 BT474 TVB-2640 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	1 nM TVB-2640
SA376277	91 BT474 TVB-2640 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	1 nM TVB-2640
SA376281	80 BT474 TVB-2640 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	200 nM TVB-2640
SA376282	87 BT474 TVB-2640 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	200 nM TVB-2640
SA376283	94 BT474 TVB-2640 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	200 nM TVB-2640
SA376278	78 BT474 TVB-2640 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	20 nM TVB-2640
SA376279	85 BT474 TVB-2640 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	20 nM TVB-2640
SA376280	92 BT474 TVB-2640 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	20 nM TVB-2640
SA376290	95 BT474 TVB-2640 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	500 nM TVB-2640
SA376291	81 BT474 TVB-2640 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	500 nM TVB-2640
SA376292	88 BT474 TVB-2640 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	500 nM TVB-2640
SA376287	79 BT474 TVB-2640 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	50 nM TVB-2640
SA376288	93 BT474 TVB-2640 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	50 nM TVB-2640
SA376289	86 BT474 TVB-2640 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	50 nM TVB-2640
SA376284	96 BT474 TVB-2640 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	5 uM TVB-2640
SA376285	89 BT474 TVB-2640 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	5 uM TVB-2640
SA376286	82 BT474 TVB-2640 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-2640	5 uM TVB-2640
SA376293	139 BT474 TVB-3166 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	1 nM TVB-3166
SA376294	132 BT474 TVB-3166 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	1 nM TVB-3166
SA376295	125 BT474 TVB-3166 1 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	1 nM TVB-3166
SA376299	142 BT474 TVB-3166 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	200 nM TVB-3166
SA376300	128 BT474 TVB-3166 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	200 nM TVB-3166
SA376301	135 BT474 TVB-3166 200 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	200 nM TVB-3166
SA376296	140 BT474 TVB-3166 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	20 nM TVB-3166
SA376297	133 BT474 TVB-3166 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	20 nM TVB-3166
SA376298	126 BT474 TVB-3166 20 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	20 nM TVB-3166
SA376308	143 BT474 TVB-3166 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	500 nM TVB-3166
SA376309	136 BT474 TVB-3166 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	500 nM TVB-3166
SA376310	129 BT474 TVB-3166 500 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	500 nM TVB-3166
SA376305	134 BT474 TVB-3166 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	50 nM TVB-3166
SA376306	127 BT474 TVB-3166 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	50 nM TVB-3166
SA376307	141 BT474 TVB-3166 50 nM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	50 nM TVB-3166
SA376302	130 BT474 TVB-3166 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	5 uM TVB-3166
SA376303	144 BT474 TVB-3166 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	5 uM TVB-3166
SA376304	137 BT474 TVB-3166 5 uM cells	BT-474 Breast cancer cells	Intracellular	TVB-3166	5 uM TVB-3166
SA376374	05 BT474 Cerulenin 0.5 uM med	BT-474 Breast cancer cells	Medium	cerulenin	0.5 uM cerulenin
SA376375	13 BT474 Cerulenin 0.5 uM med	BT-474 Breast cancer cells	Medium	cerulenin	0.5 uM cerulenin
SA376376	21 BT474 Cerulenin 0.5 uM med	BT-474 Breast cancer cells	Medium	cerulenin	0.5 uM cerulenin
SA376377	14 BT474 Cerulenin 2.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	2.0 uM cerulenin
SA376378	22 BT474 Cerulenin 2.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	2.0 uM cerulenin
SA376379	06 BT474 Cerulenin 2.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	2.0 uM cerulenin
SA376380	26 BT474 Cerulenin 20 uM med	BT-474 Breast cancer cells	Medium	cerulenin	20 uM cerulenin
SA376381	10 BT474 Cerulenin 20 uM med	BT-474 Breast cancer cells	Medium	cerulenin	20 uM cerulenin
SA376382	18 BT474 Cerulenin 20 uM med	BT-474 Breast cancer cells	Medium	cerulenin	20 uM cerulenin
SA376383	07 BT474 Cerulenin 4.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	4.0 uM cerulenin
SA376384	23 BT474 Cerulenin 4.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	4.0 uM cerulenin
SA376385	15 BT474 Cerulenin 4.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	4.0 uM cerulenin
SA376386	27 BT474 Cerulenin 50 uM med	BT-474 Breast cancer cells	Medium	cerulenin	50 uM cerulenin
SA376387	11 BT474 Cerulenin 50 uM med	BT-474 Breast cancer cells	Medium	cerulenin	50 uM cerulenin
SA376388	19 BT474 Cerulenin 50 uM med	BT-474 Breast cancer cells	Medium	cerulenin	50 uM cerulenin
SA376389	09 BT474 Cerulenin 6.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	6.0 uM cerulenin
SA376390	25 BT474 Cerulenin 6.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	6.0 uM cerulenin
SA376391	17 BT474 Cerulenin 6.0 uM med	BT-474 Breast cancer cells	Medium	cerulenin	6.0 uM cerulenin
SA376329	108 BT474 TVB-3166 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376330	116 BT474 TVB-3166 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376331	60 BT474 TVB-2640 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376332	68 BT474 TVB-2640 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376333	12 BT474 Cerulenin 0 uM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376334	20 BT474 Cerulenin 0 uM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376335	04 BT474 Cerulenin 0 uM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376336	52 BT474 TVB-2640 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376337	100 BT474 TVB-3166 0 nM med	BT-474 Breast cancer cells	Medium	NA	Control
SA376338	53 BT474 TVB-2640 1 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	1 nM TVB-2640
SA376339	61 BT474 TVB-2640 1 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	1 nM TVB-2640
SA376340	69 BT474 TVB-2640 1 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	1 nM TVB-2640
SA376344	57 BT474 TVB-2640 200 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	200 nM TVB-2640
SA376345	65 BT474 TVB-2640 200 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	200 nM TVB-2640
SA376346	73 BT474 TVB-2640 200 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	200 nM TVB-2640
SA376341	62 BT474 TVB-2640 20 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	20 nM TVB-2640
SA376342	54 BT474 TVB-2640 20 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	20 nM TVB-2640
SA376343	70 BT474 TVB-2640 20 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	20 nM TVB-2640
SA376353	58 BT474 TVB-2640 500 nM med	BT-474 Breast cancer cells	Medium	TVB-2640	500 nM TVB-2640

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Collection:

Collection ID:	CO003531
Collection Summary:	For medium metabolites, 100 µl of medium was mixed with 400 µl of 100% methanol (80% final). 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:	TR003547
Treatment Summary:	Cells were grown in RPMI-1640 supplemented with 10% dialyzed FBS and treated with various drug concentrations - cerulenin, TVB-2640, and TVB-3166 for 24 h.
Treatment Compound:	Cerulenin, TVB-2640 (Denifanstat), and TVB-3166
Treatment Doseduration:	24 h
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:	SP003545
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	AN005604	AN005605
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:	CH004258
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:	MS005329
Analysis ID:	AN005604
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
Source Temperature:	500 °C
Spray Voltage:	-4500

MS ID:	MS005330
Analysis ID:	AN005605
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Data were analyzed in 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
Source Temperature:	500 °C
Spray Voltage:	4500