Summary of study ST001033

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 PR000691. The data can be accessed directly via it's Project DOI: 10.21228/M8C10N This work is supported by NIH grant, U2C- DK119886.

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Study IDST001033
Study TitleDetermination of mode of action of anti-malalrial drugs using untargeted metabolomics
Study SummaryThe mode of action of a representative active compound was investigated using an unbiased metabolomics approach, which has previously been shown to reveal both novel and established modes of action of antimalarials (Creek et al 2016, DOI: 10.1128/AAC.01226-16). The active antimalarial OSM-S-313, and the inactive analogue OSM-S-291, were incubated with trophozoite stage P. falciparum parasites for five hours alongside reference compounds including atovaquone (ATV), chloroquine (CQ), dihydroartemisisin (DHA) and three PfATP4 inhibitors, MMV00073, MMV397264 and MMV390482. Metabolomics analysis of cell pellets and spent media allowed reproducible detection of diverse metabolites from a range of metabolic pathways, with the most significant OSM-S-313-induced perturbations observed within peptide, lipid and energy metabolism, suggesting a specific impact on parasite metabolism.
Institute
Monash University
Last NameCreek
First NameDarren
AddressMonash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Emaildarren.creek@monash.edu
Phone+61 (0) 3 9903 9249
Submit Date2018-08-13
Raw Data AvailableYes
Analysis Type DetailLC-MS
Release Date2018-08-27
Release Version1
Darren Creek Darren Creek
https://dx.doi.org/10.21228/M8C10N
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000691
Project DOI:doi: 10.21228/M8C10N
Project Title:A Potent, in vivo Active Antimalarial Series Based on a Triazolopyrazine Core: Open Source Malaria Series 4
Project Summary:The mode of action of a representative active compound was investigated using an unbiased metabolomics approach, which has previously been shown to reveal both novel and established modes of action of antimalarials (Creek et al 2016, DOI: 10.1128/AAC.01226-16). The active antimalarial OSM-S-313, and the inactive analogue OSM-S-291, were incubated with trophozoite stage P. falciparum parasites for five hours alongside reference compounds including atovaquone (ATV), chloroquine (CQ), dihydroartemisisin (DHA) and three PfATP4 inhibitors, MMV00073, MMV397264 and MMV390482. Metabolomics analysis of cell pellets and spent media allowed reproducible detection of diverse metabolites from a range of metabolic pathways, with the most significant OSM-S-313-induced perturbations observed within peptide, lipid and energy metabolism, suggesting a specific impact on parasite metabolism.
Institute:Monash University
Last Name:Creek
First Name:Darren
Address:Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Email:darren.creek@monash.edu
Phone:+61 (0) 3 9903 9249

Subject:

Subject ID:SU001072
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:36329
Genotype Strain:3D7
Subject Comments:Malaria parasite

Factors:

Subject type: Cultured cells; Subject species: Plasmodium falciparum (Factor headings shown in green)

mb_sample_id local_sample_id drug treatment
SA069162P_ATV_3Atovaquone
SA069163P_ATV_1Atovaquone
SA069164P_ATV_2Atovaquone
SA069165P_ATV_4Atovaquone
SA069166P_CQ_2Chloroquine
SA069167P_CQ_4Chloroquine
SA069168P_CQ_1Chloroquine
SA069169P_CQ_3Chloroquine
SA069174P_DHA_2Dihydroartemisisin
SA069175P_DHA_4Dihydroartemisisin
SA069176P_DHA_1Dihydroartemisisin
SA069177P_DHA_3Dihydroartemisisin
SA069170P_DMSO_1DMSO
SA069171P_DMSO_3DMSO
SA069172P_DMSO_2DMSO
SA069173P_DMSO_4DMSO
SA069178P_000073_2MMV00073
SA069179P_000073_4MMV00073
SA069180P_000073_3MMV00073
SA069181P_000073_1MMV00073
SA069182P_390482_3MMV390482
SA069183P_390482_4MMV390482
SA069184P_390482_2MMV390482
SA069185P_390482_1MMV390482
SA069186P_397264_3MMV397264
SA069187P_397264_4MMV397264
SA069188P_397264_1MMV397264
SA069189P_397264_2MMV397264
SA069190P_S291_3OSM-S-291
SA069191P_S291_4OSM-S-291
SA069192P_S291_2OSM-S-291
SA069193P_S291_1OSM-S-291
SA069194P_S313_4OSM-S-313
SA069195P_S313_2OSM-S-313
SA069196P_S313_1OSM-S-313
SA069197P_S313_3OSM-S-313
Showing results 1 to 36 of 36

Collection:

Collection ID:CO001066
Collection Summary:Plasmodium falciparum (3D7 strain) parasites were cultured in vitro according to the established method (Trager and Jensen 1976, DOI: 10.1126/science.781840) with minor modifications and incubated with test compounds as previously described (Creek et al 2016, DOI: 10.1128/AAC.01226-16). Briefly, parasites were brought to a tightly synchronous life stage population (within 4 hours of the 48 hour life cycle) by treating with 5% (w/v) sorbitol twice at an interval of 14 hours, and incubated for a further 58 hours to bring all parasites to mid-trophozoite stage (27-31 hours post infection). These cells were used for drug treatment and further analysis.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR001086
Treatment Summary:In 96 well plates, 200 μl cultures at 7% parasitemia and 3% haematocrit were incubated with 1 µM of test compounds for a further 5 hours (32-36 hours post infection). Each compound was incubated in four replicates and untreated controls were treated with DMSO.

Sample Preparation:

Sampleprep ID:SP001079
Sampleprep Summary:After incubation with the test compounds for 5 hours, all red blood cells were settled at the bottom of the culture wells. Culture medium was carefully removed and the metabolism of the cells was quenched by placing the plate on ice and adding ice-cold phosphate buffered saline (PBS) to the culture wells. All subsequent extraction steps were performed on ice. Cells were centrifuged for 5 min at 400g in a chilled centrifuge at 4°C. The supernatant was carefully removed and 135 µl of ice-cold methanol (containing internal standards TRIS, CHAPS, CAPS and PIPES) was added followed by rapid mixing of the cell suspension using the pipette three times. Spent media samples were also prepared by adding 10 µl of the culture supernatants to 140 µl of ice-cold methanol (containing internal standards). All samples were agitated on ice for 1 hour and then centrifuged for 10 min at 1000g in a chilled centrifuge at 4°C. The supernatant was transferred to glass vials and stored at -80°C until analysis.

Combined analysis:

Analysis ID AN001694
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS
Column ZIC-pHILIC (Merck Sequant) column
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units Peak height

Chromatography:

Chromatography ID:CH001193
Chromatography Summary:Metabolite extracts were analysed using hydrophilic interaction (HILIC) liquid chromatography (LC) and high resolution mass spectrometry on an Orbitrap system.
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:ZIC-pHILIC (Merck Sequant) column
Column Temperature:25°C
Flow Gradient:linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%.
Flow Rate:300 μL/min
Internal Standard:internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM)
Sample Injection:10 μL
Solvent A:20 mM ammonium carbonate in water
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS001569
Analysis ID:AN001694
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:UNSPECIFIED
Capillary Temperature:300°C
Capillary Voltage:+50 V
Spray Voltage:4kV
Resolution Setting:35000
Scan Range Moverz:85- 1275 m/z
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