Summary of study ST001205

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 IDST001205
Study TitlePeroxide antimalarial treatment of K13-mutant and -wildtype P. falciparum parasites
Study SummaryRed blood cells (RBCs) infected with trophozoite stage P. falciparum parasites (Cam3.IIR539T or Cam3.IIrev lines) at 4% parasitaemia and 2% haematocrit were treated with 100 nM of DHA, OZ277 or OZ439 for a duration of 1, 3 and 5 h, respectively. The K13-mutant artemisinin resistant parasite line used was Cam3.IIR539T. The K13-wildtype artemisinin sensitive parasite line used was Cam3.IIrev. The Samples treated with vehicle (DMSO) acted as the untreated control.
Institute
Monash University
Last NameGiannangelo
First NameCarlo
Address381 Royal Parade, Parkville, Victoria, 3052, Australia
Emailcarlo.giannangelo@monash.edu
Phone99039282
Submit Date2019-06-26
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:SU001272
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum;Homo sapiens
Taxonomy ID:5833;9606
Genotype Strain:Cam3.IIR539T and Cam3.IIrev
Age Or Age Range:22-26 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 parasite_line treatment treatment_duration_(h)
SA0847421h_DHA_R1_troph_dCam3.IIR539T DHA 1
SA0847431h_DHA_R1_troph_bCam3.IIR539T DHA 1
SA0847441h_DHA_R1_troph_cCam3.IIR539T DHA 1
SA0847451h_DMSO_R1_troph_dCam3.IIR539T DMSO 1
SA0847461h_DMSO_R1_troph_cCam3.IIR539T DMSO 1
SA0847475h_DMSO_R1_troph_dCam3.IIR539T DMSO 5
SA0847485h_DMSO_R1_troph_bCam3.IIR539T DMSO 5
SA0847495h_DMSO_R1_troph_cCam3.IIR539T DMSO 5
SA0847503h_OZ277_R1_troph_bCam3.IIR539T OZ277 3
SA0847513h_OZ277_R1_troph_cCam3.IIR539T OZ277 3
SA0847523h_OZ277_R1_troph_dCam3.IIR539T OZ277 3
SA0847535h_OZ439_R1_troph_bCam3.IIR539T OZ439 5
SA0847545h_OZ439_R1_troph_cCam3.IIR539T OZ439 5
SA0847555h_OZ439_R1_troph_dCam3.IIR539T OZ439 5
SA0847561h_DHA_S_troph_bCam3.IIrev DHA 1
SA0847571h_DHA_S_troph_cCam3.IIrev DHA 1
SA0847581h_DHA_S_troph_dCam3.IIrev DHA 1
SA0847591h_DMSO_S_troph_dCam3.IIrev DMSO 1
SA0847601h_DMSO_S_troph_cCam3.IIrev DMSO 1
SA0847615h_DMSO_S_troph_cCam3.IIrev DMSO 5
SA0847625h_DMSO_S_troph_bCam3.IIrev DMSO 5
SA0847635h_DMSO_S_troph_dCam3.IIrev DMSO 5
SA0847643h_OZ277_S_troph_dCam3.IIrev OZ277 3
SA0847653h_OZ277_S_troph_cCam3.IIrev OZ277 3
SA0847663h_OZ277_S_troph_eCam3.IIrev OZ277 3
SA0847675h_OZ439_S_troph_eCam3.IIrev OZ439 5
SA0847685h_OZ439_S_troph_cCam3.IIrev OZ439 5
SA0847695h_OZ439_S_troph_dCam3.IIrev OZ439 5
Showing results 1 to 28 of 28

Collection:

Collection ID:CO001266
Collection Summary:RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) were adjusted to 4% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 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 150 µL of cold methanol. 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:TR001287
Treatment Summary:RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) at 4% parasitaemia and 2% Hct were treated with 100 nM of DHA, OZ277 or OZ439 (or an equivalent volume of DMSO) for 1, 3 and 5 h, respectively. During the drug incubation period parasite cultures 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:SP001280
Sampleprep Summary:RBCs infected with Cam3.IIR539T (resistant) or Cam3.IIrev (sensitive) P. falciparum parasites (22-26 h post invasion) were adjusted to 4% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 1E8 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 150 µL of cold methanol. 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
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN002006 AN002007
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:CH001451
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:MS001859
Analysis ID:AN002006
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:MS001860
Analysis ID:AN002007
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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