Summary of study ST001175

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 PR000787. The data can be accessed directly via it's Project DOI: 10.21228/M8Z97B This work is supported by NIH grant, U2C- DK119886.

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Study IDST001175
Study TitleMulti-omics analysis demonstrates unique mode of action of a potent new antimalarial compound, JPC-3210, against Plasmodium falciparum
Study SummaryThe increasing incidence of antimalarial drug resistance to the first-line artemisinins, and their combination partner drugs, underpins an urgent need for new antimalarial drugs, ideally with a novel mechanism of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum, low cytotoxicity, potent in vivo efficacy against murine malaria, and favourable preclinical pharmacokinetics, including a lengthy plasma elimination half-life. This study demonstrates the application of a “multi-omics” workflow based on high resolution orbitrap mass spectrometry to investigate the impact of JPC-3210 on biochemical pathways within P. falciparum infected red blood cells. Metabolomics and peptidomics analysis revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a depletion in short hemoglobin-derived peptides, while peptidomics analysis showed a depletion in longer hemoglobin-derived peptides. In order to further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerisation assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerisation, about 10-fold less potent then the quinoline antimalarials. Furthermore, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature in comparison to other known antimalarials. Whilst JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. In conclusion, multi-omics studies using high resolution mass spectrometry revealed JPC-3210 to possess a unique mechanism of action involving inhibition of hemoglobin digestion, depletion of DNA replication and synthesis proteins, and elevation of regulators of protein translation. Importantly, this mechanism is distinct from currently-used antimalarials, suggesting that JPC-3210 warrants further investigation as a potentially useful new antimalarial agent.
Institute
Monash University
Last NameSiddiqui
First NameGhizal
Address381 Royal Parade, Parkville
Emailghizal.siddiqui@monash.edu
Phone99039282
Submit Date2019-04-25
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-05-15
Release Version1
Ghizal Siddiqui Ghizal Siddiqui
https://dx.doi.org/10.21228/M8Z97B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000787
Project DOI:doi: 10.21228/M8Z97B
Project Title:Multi-omics analysis demonstrates unique mode of action of a potent new antimalarial compound, JPC-3210, against Plasmodium falciparum
Project Summary:The increasing incidence of antimalarial drug resistance to the first-line artemisinins, and their combination partner drugs, underpins an urgent need for new antimalarial drugs, ideally with a novel mechanism of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum, low cytotoxicity, potent in vivo efficacy against murine malaria, and favourable preclinical pharmacokinetics, including a lengthy plasma elimination half-life. This study demonstrates the application of a “multi-omics” workflow based on high resolution orbitrap mass spectrometry to investigate the impact of JPC-3210 on biochemical pathways within P. falciparum infected red blood cells. Metabolomics and peptidomics analysis revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a depletion in short hemoglobin-derived peptides, while peptidomics analysis showed a depletion in longer hemoglobin-derived peptides. In order to further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerisation assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerisation, about 10-fold less potent then the quinoline antimalarials. Furthermore, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature in comparison to other known antimalarials. Whilst JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. In conclusion, multi-omics studies using high resolution mass spectrometry revealed JPC-3210 to possess a unique mechanism of action involving inhibition of hemoglobin digestion, depletion of DNA replication and synthesis proteins, and elevation of regulators of protein translation. Importantly, this mechanism is distinct from currently-used antimalarials, suggesting that JPC-3210 warrants further investigation as a potentially useful new antimalarial agent.
Institute:Monash University
Last Name:Siddiqui
First Name:Ghizal
Address:381 Royal Parade, Parkville
Email:ghizal.siddiqui@monash.edu
Phone:99039282

Subject:

Subject ID:SU001241
Subject Type:Other
Subject Species:Plasmodium falciparum
Taxonomy ID:5833

Factors:

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

mb_sample_id local_sample_id Condition
SA081519P_Aqm_1Aqm treated
SA081520P_Aqm_3Aqm treated
SA081521P_Aqm_2Aqm treated
SA081522P_Aqm_4Aqm treated
SA081523P_Atov_2Atov treated
SA081524P_Atov_4Atov treated
SA081525P_Atov_1Atov treated
SA081526P_Atov_3Atov treated
SA081527Blank_3Blank
SA081528Blank_2Blank
SA081529Blank_6Blank
SA081530Blank_1Blank
SA081531Blank_4Blank
SA081532Blank_5Blank
SA081533P_CQ_4CQ treated
SA081534P_CQ_3CQ treated
SA081535P_CQ_2CQ treated
SA081536P_CQ_1CQ treated
SA081537P_DHA_4DHA treated
SA081538P_DHA_3DHA treated
SA081539P_DHA_2DHA treated
SA081540P_DHA_1DHA treated
SA081541P_DMSO_6DMSO treated
SA081542P_DMSO_8DMSO treated
SA081543P_DMSO_7DMSO treated
SA081544P_DMSO_2DMSO treated
SA081545P_DMSO_3DMSO treated
SA081546P_DMSO_4DMSO treated
SA081547P_DMSO_5DMSO treated
SA081548P_JPC_3JPC treated
SA081549P_JPC_2JPC treated
SA081550P_JPC_1JPC treated
SA081551P_LF_1LF treated
SA081552P_LF_2LF treated
SA081553P_LF_4LF treated
SA081554P_LF_3LF treated
SA081555P_MQ_2MQ treated
SA081556P_MQ_1MQ treated
SA081557P_MQ_3MQ treated
SA081558P_MQ_4MQ treated
SA081559P_PYN_3PYN treated
SA081560P_PYN_4PYN treated
SA081561P_PYN_1PYN treated
SA081562P_PYN_2PYN treated
SA081563QC_P_6QC
SA081564QC_P_5QC
SA081565QC_P_3QC
SA081566QC_P_1QC
SA081567QC_P_2QC
SA081568QC_P_4QC
SA081569P_TQ_1TQ treated
SA081570P_TQ_2TQ treated
SA081571P_TQ_3TQ treated
SA081572P_TQ_4TQ treated
Showing results 1 to 54 of 54

Collection:

Collection ID:CO001235
Collection Summary:Plasmodium falciparum infected red blood cells treated with different drugs were counted washed and metabolites extracted used 100% methanol
Sample Type:Plasmodium cells

Treatment:

Treatment ID:TR001256
Treatment Summary:The metabolism of mid-trophozoite stage parasites (24-28 h post invasion) in response to treatment with a panel of antimalarial compounds was investigated. Cultures (200 μl) were adjusted to 8% parasitemia and 2% hematocrit before being exposed to test compounds; JPC-3210, AQm, PYN, ATQ, DHA, CQ, MQ, LF and TQ (1 μM) for 1 h in a flat bottom 96 well plate. At least three replicates of each compound and seven replicates of an untreated control which contained an equivalent volume of DMSO vehicle (0.01% final concentration) were analysed.

Sample Preparation:

Sampleprep ID:SP001249
Sampleprep Summary:Following drug incubation, metabolites were extracted as detailed. In short, after incubation with drug, culture medium was aspirated from each well, and the metabolism of infected red blood cells (iRBCs) was quenched by the addition of ice-cold phosphate-buffered saline (PBS). Cells were pelleted by centrifugation for 5 min at 1,000 × g, and the PBS supernatant was removed prior to the addition of 135 μL ice cold methanol (containing the internal standard compounds; CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS [N-cyclohexyl-3-aminopropane-sulfonic acid], and PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]). Samples were rapidly mixed by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 min and then centrifuged at 3,000 × g to remove insoluble material. Supernatants were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80°C until analysis. An aliquot (10 μL) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor downstream sample stability and analytical reproducibility.

Combined analysis:

Analysis ID AN001950 AN001951
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column ZIC-pHILIC column (5 μm particle size, 4.6 by 150 mm; Merck) ZIC-pHILIC column (5 μm particle size, 4.6 by 150 mm; Merck)
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 Signal Intensity Signal Intensity

Chromatography:

Chromatography ID:CH001414
Chromatography Summary:Briefly, samples (10 μL) were injected onto a Dionex RSLC U3000 LC system (Thermo) fitted with a ZIC-pHILIC column (5 μm particle size, 4.6 by 150 mm; Merck) and 20 mM ammonium carbonate (A) and acetonitrile (B) were used as the mobile phases. A 30 min gradient starting from 80% B to 40% B over 20 min, followed by washing at 5% B for 3 min and re-equilibration at 80% B, was used.
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:ZIC-pHILIC column (5 μm particle size, 4.6 by 150 mm; Merck)
Chromatography Type:HILIC

MS:

MS ID:MS001805
Analysis ID:AN001950
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:-
Ion Mode:POSITIVE
  
MS ID:MS001806
Analysis ID:AN001951
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:-
Ion Mode:NEGATIVE
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