Summary of study ST001202

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 PR000809. The data can be accessed directly via it's Project DOI: 10.21228/M83X38 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001202
Study TitlePeroxide antimalarial treatment timecourse on ring-stage P. falciparum parasites
Study SummaryRed blood cells (RBCs) infected with ring stage P. falciparum parasites (3D7 strain) at 10% parasitaemia and 2% haematocrit were treated with OZ277 (1 uM), OZ439 (1 uM), DHA (300 nM) or vehicle (0.03% DMSO). This was a 5-timepoint study, with samples taken 0, 1.5, 3, 6 and 9 h after drug or vehicle addition. Samples treated with vehicle acted as the untreated control. Samples from drug treated uninfected RBCs were also taken to ensure the observed drug effects were parasite specific.
Institute
Monash University
Last NameGiannangelo
First NameCarlo
Address381 Royal Parade, Parkville, Victoria, 3052, Australia
Emailcarlo.giannangelo@monash.edu
Phone99039282
Submit Date2019-06-24
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-07-17
Release Version1
Carlo Giannangelo Carlo Giannangelo
https://dx.doi.org/10.21228/M83X38
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000809
Project DOI:doi: 10.21228/M83X38
Project Title:System-wide biochemical analysis reveals ozonide and artemisinin antimalarials initially act by disrupting malaria parasite haemoglobin digestion
Project Summary:Artemisinins are currently the first-line antimalarials, and rely on a peroxide pharmacophore for their potent activity. OZ277 (arterolane) and OZ439 (artefenomel) are newer synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a “multi-omics” workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of peroxide-treated P. falciparum infected red blood cells (iRBCs) revealed a rapid depletion of short Hb-derived peptides, while untargeted peptidomics showed accumulation of longer Hb peptides. Quantitative proteomics and ABPP assays demonstrated that Hb digesting proteases were significantly increased in abundance and activity following treatment, respectively. The association between peroxide activity and Hb catabolism was also confirmed in a K13-mutant artemisinin resistant parasite line. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in peroxide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short peroxide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate peroxide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage peroxide-induced damage.
Institute:Monash University
Last Name:Giannangelo
First Name:Carlo
Address:381 Royal Parade, Parkville, Victoria, 3052, Australia
Email:carlo.giannangelo@monash.edu
Phone:99039282

Subject:

Subject ID:SU001269
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum;Homo sapiens
Taxonomy ID:5833/9606
Genotype Strain:3D7
Age Or Age Range:6-12 h post invasion

Factors:

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

mb_sample_id local_sample_id cell_type treatment treatment_duration_(h)
SA084070DHA_i0h_aiRBC DHA -
SA084071DHA_i0h_diRBC DHA -
SA084072DHA_i0h_biRBC DHA -
SA084073DHA_i0h_ciRBC DHA -
SA084074DHA_i1_5h_ciRBC DHA 1.5
SA084075DHA_i1_5h_biRBC DHA 1.5
SA084076DHA_i1_5h_diRBC DHA 1.5
SA084077DHA_i1_5h_aiRBC DHA 1.5
SA084078DHA_i3h_ciRBC DHA 3
SA084079DHA_i3h_biRBC DHA 3
SA084080DHA_i3h_aiRBC DHA 3
SA084081DHA_i3h_diRBC DHA 3
SA084082DHA_i6h_aiRBC DHA 6
SA084083DHA_i6h_biRBC DHA 6
SA084084DHA_i6h_ciRBC DHA 6
SA084085DHA_i6h_diRBC DHA 6
SA084086DMSO_i0h_diRBC DMSO -
SA084087DMSO_i0h_biRBC DMSO -
SA084088DMSO_i0h_aiRBC DMSO -
SA084089DMSO_i0h_ciRBC DMSO -
SA084090DMSO_i1_5h_aiRBC DMSO 1.5
SA084091DMSO_i1_5h_diRBC DMSO 1.5
SA084092DMSO_i1_5h_biRBC DMSO 1.5
SA084093DMSO_i1_5h_ciRBC DMSO 1.5
SA084094DMSO_i3h_ciRBC DMSO 3
SA084095DMSO_i3h_biRBC DMSO 3
SA084096DMSO_i3h_diRBC DMSO 3
SA084097DMSO_i3h_aiRBC DMSO 3
SA084098DMSO_i6h_diRBC DMSO 6
SA084099DMSO_i6h_aiRBC DMSO 6
SA084100DMSO_i6h_ciRBC DMSO 6
SA084101DMSO_i6h_biRBC DMSO 6
SA084102DMSO_i9h_ciRBC DMSO 9
SA084103DMSO_i9h_diRBC DMSO 9
SA084104DMSO_i9h_biRBC DMSO 9
SA084105DMSO_i9h_aiRBC DMSO 9
SA084106OZ277_i0h_aiRBC OZ277 -
SA084107OZ277_i0h_biRBC OZ277 -
SA084108OZ277_i0h_diRBC OZ277 -
SA084109OZ277_i0h_ciRBC OZ277 -
SA084110OZ277_i3h_diRBC OZ277 3
SA084111OZ277_i3h_biRBC OZ277 3
SA084112OZ277_i3h_ciRBC OZ277 3
SA084113OZ277_i3h_aiRBC OZ277 3
SA084114OZ277_i6h_aiRBC OZ277 6
SA084115OZ277_i6h_ciRBC OZ277 6
SA084116OZ277_i6h_diRBC OZ277 6
SA084117OZ277_i6h_biRBC OZ277 6
SA084118OZ277_i9h_diRBC OZ277 9
SA084119OZ277_i9h_ciRBC OZ277 9
SA084120OZ277_i9h_aiRBC OZ277 9
SA084121OZ277_i9h_biRBC OZ277 9
SA084122OZ439_i0h_aiRBC OZ439 -
SA084123OZ439_i0h_ciRBC OZ439 -
SA084124OZ439_i0h_diRBC OZ439 -
SA084125OZ439_i0h_biRBC OZ439 -
SA084126OZ439_i3h_aiRBC OZ439 3
SA084127OZ439_i3h_ciRBC OZ439 3
SA084128OZ439_i3h_biRBC OZ439 3
SA084129OZ439_i3h_diRBC OZ439 3
SA084130OZ439_i6h_ciRBC OZ439 6
SA084131OZ439_i6h_aiRBC OZ439 6
SA084132OZ439_i6h_biRBC OZ439 6
SA084133OZ439_i6h_diRBC OZ439 6
SA084134OZ439_i9h_ciRBC OZ439 9
SA084135OZ439_i9h_aiRBC OZ439 9
SA084136OZ439_i9h_biRBC OZ439 9
SA084137OZ439_i9h_diRBC OZ439 9
SA084138DHA_u0h_dunRBC DHA -
SA084139DHA_u0h_bunRBC DHA -
SA084140DHA_u0h_cunRBC DHA -
SA084141DHA_u0h_aunRBC DHA -
SA084142DHA_u1_5h_bunRBC DHA 1.5
SA084143DHA_u1_5h_cunRBC DHA 1.5
SA084144DHA_u1_5h_aunRBC DHA 1.5
SA084145DHA_u1_5h_dunRBC DHA 1.5
SA084146DHA_u3h_cunRBC DHA 3
SA084147DHA_u3h_aunRBC DHA 3
SA084148DHA_u3h_bunRBC DHA 3
SA084149DHA_u3h_dunRBC DHA 3
SA084150DHA_u6h_dunRBC DHA 6
SA084151DHA_u6h_aunRBC DHA 6
SA084152DHA_u6h_bunRBC DHA 6
SA084153DHA_u6h_cunRBC DHA 6
SA084154DMSO_u0h_dunRBC DMSO -
SA084155DMSO_u0h_cunRBC DMSO -
SA084156DMSO_u0h_aunRBC DMSO -
SA084157DMSO_u0h_bunRBC DMSO -
SA084158DMSO_u1_5h_aunRBC DMSO 1.5
SA084159DMSO_u1_5h_dunRBC DMSO 1.5
SA084160DMSO_u1_5h_cunRBC DMSO 1.5
SA084161DMSO_u1_5h_bunRBC DMSO 1.5
SA084162DMSO_u3h_dunRBC DMSO 3
SA084163DMSO_u3h_aunRBC DMSO 3
SA084164DMSO_u3h_bunRBC DMSO 3
SA084165DMSO_u3h_cunRBC DMSO 3
SA084166DMSO_u6h_bunRBC DMSO 6
SA084167DMSO_u6h_dunRBC DMSO 6
SA084168DMSO_u6h_aunRBC DMSO 6
SA084169DMSO_u6h_cunRBC DMSO 6
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Collection:

Collection ID:CO001263
Collection Summary:Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 2E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 200 µL of cold chloroform/methanol/water (1:3:1). The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR001284
Treatment Summary:Ring stage P. falciparum infected RBCs (10% parasitaemia and 2% Hct) were treated with OZ277 (1 uM), OZ439 (1 uM), DHA (300 nM) or an equivalent volume of DMSO (0.03%). DHA-treated cultures were incubated with drug for 0, 1.5, 3 and 6 h. OZ277- and OZ439-treated cultures were incubated with drug for 0, 3, 6 and 9 h. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2.
Treatment Compound:OZ277 (arterolane), OZ439 (artefenomel) and dihydroartemisinin (DHA)
Treatment Vehicle:DMSO
Cell Media:Complete RPMI medium (10.4 g/L) containing HEPES (5.94 g/L), hypoxanthine (50 mg/L), sodium bicarbonate (2.1 g/L) and Albumax II (5 g/L).
Cell Media Lastchanged:Immediately prior to initiation of drug incubation

Sample Preparation:

Sampleprep ID:SP001277
Sampleprep Summary:Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 2E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 200 µL of cold chloroform/methanol/water (1:3:1). The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.
Processing Storage Conditions:Described in summary

Combined analysis:

Analysis ID AN002000 AN002001
Analysis type MS MS
Chromatography type pHILIC pHILIC
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Peak intensity Peak intensity

Chromatography:

Chromatography ID:CH001447
Chromatography Summary:The 32 min gradient HPLC run was from 80% B to 50% B over 15 min, then to 5% B at 18 min, followed by a wash with 5% B for 3 min and re-equilibrated with 80% B at a flow rate of 0.3 mL/min.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
Solvent A:20 mM ammonium carbonate
Solvent B:100% acetonitrile
Chromatography Type:pHILIC

MS:

MS ID:MS001853
Analysis ID:AN002000
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:POSITIVE
  
MS ID:MS001854
Analysis ID:AN002001
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times.
Ion Mode:NEGATIVE
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