Summary of Study ST001201

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 IDST001201
Study TitlePeroxide antimalarial treatment timecourse on trophozoite-stage P. falciparum parasites
Study SummaryRed blood cells (RBCs) infected with trophozoite stage P. falciparum parasites (3D7 strain) at 10% parasitaemia and 2% haematocrit were treated with OZ277 (300 nM), OZ439 (300 nM), DHA (100 nM) or vehicle (0.03% DMSO). This was a 4-timepoint study, with samples taken 0, 0.5, 1.5 and 3 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 Date2019-06-19
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
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:SU001268
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum;Homo sapiens
Taxonomy ID:5833;9606
Genotype Strain:3D7
Age Or Age Range:28-34 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)
SA0838780h_DHA_iRBC_bbiRBC DHA -
SA0838790h_DHA_iRBC_biRBC DHA -
SA0838800h_DHA_iRBC_aiRBC DHA -
SA0838810h_DHA_iRBC_diRBC DHA -
SA0838820h_DHA_iRBC_ddiRBC DHA -
SA0838830h_DHA_iRBC_aaiRBC DHA -
SA0838840h_DHA_iRBC_cciRBC DHA -
SA0838850h_DHA_iRBC_ciRBC DHA -
SA0838860_5h_DHA_iRBC_ciRBC DHA 0.5
SA0838870_5h_DHA_iRBC_aaiRBC DHA 0.5
SA0838880_5h_DHA_iRBC_bbiRBC DHA 0.5
SA0838890_5h_DHA_iRBC_biRBC DHA 0.5
SA0838900_5h_DHA_iRBC_aiRBC DHA 0.5
SA0838910_5h_DHA_iRBC_diRBC DHA 0.5
SA0838920_5h_DHA_iRBC_cciRBC DHA 0.5
SA0838930_5h_DHA_iRBC_ddiRBC DHA 0.5
SA0838941_5h_DHA_iRBC_aaiRBC DHA 1.5
SA0838951_5h_DHA_iRBC_bbiRBC DHA 1.5
SA0838961_5h_DHA_iRBC_cciRBC DHA 1.5
SA0838971_5h_DHA_iRBC_aiRBC DHA 1.5
SA0838981_5h_DHA_iRBC_ddiRBC DHA 1.5
SA0838991_5h_DHA_iRBC_ciRBC DHA 1.5
SA0839001_5h_DHA_iRBC_biRBC DHA 1.5
SA0839011_5h_DHA_iRBC_diRBC DHA 1.5
SA0839023h_DHA_iRBC_ddiRBC DHA 3
SA0839033h_DHA_iRBC_ciRBC DHA 3
SA0839043h_DHA_iRBC_aaiRBC DHA 3
SA0839053h_DHA_iRBC_cciRBC DHA 3
SA0839063h_DHA_iRBC_biRBC DHA 3
SA0839073h_DHA_iRBC_bbiRBC DHA 3
SA0839083h_DHA_iRBC_aiRBC DHA 3
SA0839093h_DHA_iRBC_diRBC DHA 3
SA0839100h_DMSO_iRBC_aiRBC DMSO -
SA0839110h_DMSO_iRBC_biRBC DMSO -
SA0839120h_DMSO_iRBC_bbiRBC DMSO -
SA0839130h_DMSO_iRBC_cciRBC DMSO -
SA0839140h_DMSO_iRBC_ciRBC DMSO -
SA0839150h_DMSO_iRBC_aaiRBC DMSO -
SA0839160h_DMSO_iRBC_diRBC DMSO -
SA0839170h_DMSO_iRBC_ddiRBC DMSO -
SA0839180_5h_DMSO_iRBC_ciRBC DMSO 0.5
SA0839190_5h_DMSO_iRBC_aaiRBC DMSO 0.5
SA0839200_5h_DMSO_iRBC_aiRBC DMSO 0.5
SA0839210_5h_DMSO_iRBC_diRBC DMSO 0.5
SA0839220_5h_DMSO_iRBC_biRBC DMSO 0.5
SA0839230_5h_DMSO_iRBC_cciRBC DMSO 0.5
SA0839240_5h_DMSO_iRBC_ddiRBC DMSO 0.5
SA0839250_5h_DMSO_iRBC_bbiRBC DMSO 0.5
SA0839261_5h_DMSO_iRBC_aaiRBC DMSO 1.5
SA0839271_5h_DMSO_iRBC_aiRBC DMSO 1.5
SA0839281_5h_DMSO_iRBC_cciRBC DMSO 1.5
SA0839291_5h_DMSO_iRBC_diRBC DMSO 1.5
SA0839301_5h_DMSO_iRBC_bbiRBC DMSO 1.5
SA0839311_5h_DMSO_iRBC_biRBC DMSO 1.5
SA0839321_5h_DMSO_iRBC_ddiRBC DMSO 1.5
SA0839331_5h_DMSO_iRBC_ciRBC DMSO 1.5
SA0839343h_DMSO_iRBC_cciRBC DMSO 3
SA0839353h_DMSO_iRBC_ciRBC DMSO 3
SA0839363h_DMSO_iRBC_diRBC DMSO 3
SA0839373h_DMSO_iRBC_biRBC DMSO 3
SA0839383h_DMSO_iRBC_bbiRBC DMSO 3
SA0839393h_DMSO_iRBC_ddiRBC DMSO 3
SA0839403h_DMSO_iRBC_aiRBC DMSO 3
SA0839413h_DMSO_iRBC_aaiRBC DMSO 3
SA0839420h_OZ277_iRBC_biRBC OZ277 -
SA0839430h_OZ277_iRBC_aiRBC OZ277 -
SA0839440h_OZ277_iRBC_diRBC OZ277 -
SA0839450h_OZ277_iRBC_ciRBC OZ277 -
SA0839460_5h_OZ277_iRBC_diRBC OZ277 0.5
SA0839470_5h_OZ277_iRBC_ciRBC OZ277 0.5
SA0839480_5h_OZ277_iRBC_biRBC OZ277 0.5
SA0839490_5h_OZ277_iRBC_aiRBC OZ277 0.5
SA0839501_5h_OZ277_iRBC_biRBC OZ277 1.5
SA0839511_5h_OZ277_iRBC_diRBC OZ277 1.5
SA0839521_5h_OZ277_iRBC_ciRBC OZ277 1.5
SA0839531_5h_OZ277_iRBC_aiRBC OZ277 1.5
SA0839543h_OZ277_iRBC_biRBC OZ277 3
SA0839553h_OZ277_iRBC_ciRBC OZ277 3
SA0839563h_OZ277_iRBC_aiRBC OZ277 3
SA0839573h_OZ277_iRBC_diRBC OZ277 3
SA0839580h_OZ439_iRBC_diRBC OZ439 -
SA0839590h_OZ439_iRBC_biRBC OZ439 -
SA0839600h_OZ439_iRBC_ciRBC OZ439 -
SA0839610h_OZ439_iRBC_aiRBC OZ439 -
SA0839620_5h_OZ439_iRBC_diRBC OZ439 0.5
SA0839630_5h_OZ439_iRBC_biRBC OZ439 0.5
SA0839640_5h_OZ439_iRBC_ciRBC OZ439 0.5
SA0839650_5h_OZ439_iRBC_aiRBC OZ439 0.5
SA0839661_5h_OZ439_iRBC_ciRBC OZ439 1.5
SA0839671_5h_OZ439_iRBC_aiRBC OZ439 1.5
SA0839681_5h_OZ439_iRBC_diRBC OZ439 1.5
SA0839691_5h_OZ439_iRBC_biRBC OZ439 1.5
SA0839703h_OZ439_iRBC_aiRBC OZ439 3
SA0839713h_OZ439_iRBC_biRBC OZ439 3
SA0839723h_OZ439_iRBC_diRBC OZ439 3
SA0839733h_OZ439_iRBC_ciRBC OZ439 3
SA0839740h_DHA_unRBC_ccunRBC DHA -
SA0839750h_DHA_unRBC_aaunRBC DHA -
SA0839760h_DHA_unRBC_bunRBC DHA -
SA0839770h_DHA_unRBC_aunRBC DHA -
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Collection:

Collection ID:CO001262
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, 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 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:TR001283
Treatment Summary:Trophozoite stage P. falciparum infected RBCs (10% parasitaemia and 2% Hct) were treated with OZ277 (300 nM), OZ439 (300 nM), DHA (100 nM) or an equivalent volume of DMSO (0.03%) for 0, 0.5, 1.5 and 3 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:SP001276
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, 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 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 AN001998 AN001999
Analysis type MS MS
Chromatography type HILIC HILIC
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:CH001446
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:100% water; 20 mM ammonium carbonate
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS001851
Analysis ID:AN001998
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. 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:MS001852
Analysis ID:AN001999
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. 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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