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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001033
Study Title	Determination of mode of action of anti-malalrial drugs using untargeted metabolomics
Study 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
Submit Date	2018-08-13
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2018-08-27
Release Version	1

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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:	5833
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
SA069162	P_ATV_3	Atovaquone
SA069163	P_ATV_1	Atovaquone
SA069164	P_ATV_2	Atovaquone
SA069165	P_ATV_4	Atovaquone
SA069166	P_CQ_2	Chloroquine
SA069167	P_CQ_4	Chloroquine
SA069168	P_CQ_1	Chloroquine
SA069169	P_CQ_3	Chloroquine
SA069174	P_DHA_2	Dihydroartemisisin
SA069175	P_DHA_4	Dihydroartemisisin
SA069176	P_DHA_1	Dihydroartemisisin
SA069177	P_DHA_3	Dihydroartemisisin
SA069170	P_DMSO_1	DMSO
SA069171	P_DMSO_3	DMSO
SA069172	P_DMSO_2	DMSO
SA069173	P_DMSO_4	DMSO
SA069178	P_000073_2	MMV00073
SA069179	P_000073_4	MMV00073
SA069180	P_000073_3	MMV00073
SA069181	P_000073_1	MMV00073
SA069182	P_390482_3	MMV390482
SA069183	P_390482_4	MMV390482
SA069184	P_390482_2	MMV390482
SA069185	P_390482_1	MMV390482
SA069186	P_397264_3	MMV397264
SA069187	P_397264_4	MMV397264
SA069188	P_397264_1	MMV397264
SA069189	P_397264_2	MMV397264
SA069190	P_S291_3	OSM-S-291
SA069191	P_S291_4	OSM-S-291
SA069192	P_S291_2	OSM-S-291
SA069193	P_S291_1	OSM-S-291
SA069194	P_S313_4	OSM-S-313
SA069195	P_S313_2	OSM-S-313
SA069196	P_S313_1	OSM-S-313
SA069197	P_S313_3	OSM-S-313

Showing results 1 to 36 of 36

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.

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)
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 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:	100% water; 20 mM ammonium carbonate
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