Summary of Study ST002108

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 PR001335. The data can be accessed directly via it's Project DOI: 10.21228/M85416 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002108
Study TitleGenetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway (Part 3)
Study SummaryPlasmodium falciparum, the causative agent of malaria, continues to remain a global health threat since these parasites are now resistant to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.
Institute
Monash University
Last NameSiddiqui
First NameGhizal
Address381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Emailghizal.siddiqui@monash.edu
Phone99039282
Submit Date2022-03-17
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-04-04
Release Version1
Ghizal Siddiqui Ghizal Siddiqui
https://dx.doi.org/10.21228/M85416
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001335
Project DOI:doi: 10.21228/M85416
Project Title:Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway
Project Summary:Plasmodium falciparum, the causative agent of malaria, continues to remain a global health threat since these parasites are now resistant to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.
Institute:Monash University
Last Name:Siddiqui
First Name:Ghizal
Address:381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Email:ghizal.siddiqui@monash.edu
Phone:99039282

Subject:

Subject ID:SU002193
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833

Factors:

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

mb_sample_id local_sample_id cell_type treatment
SA20234871_irbcs_p_1iRBC Compound 3
SA20234971_irbcs_p_2iRBC Compound 3
SA20235071_irbcs_p_4iRBC Compound 3
SA20235171_irbcs_p_3iRBC Compound 3
SA202352DMSO_iRBC_p_7iRBC DMSO
SA202353DMSO_iRBC_p_8iRBC DMSO
SA202354DMSO_iRBC_p_6iRBC DMSO
SA202355DMSO_iRBC_p_9iRBC DMSO
SA202356DMSO_iRBC_p_3iRBC DMSO
SA202357DMSO_iRBC_p_1iRBC DMSO
SA202358DMSO_iRBC_p_2iRBC DMSO
SA202359DMSO_iRBC_p_4iRBC DMSO
SA202360DMSO_iRBC_p_5iRBC DMSO
Showing results 1 to 13 of 13

Collection:

Collection ID:CO002186
Collection Summary:Pf3D7 cultures underwent double sorbitol synchronization 14 h apart, followed by further incubation for 28-42 h to achieve the desired trophozoite stage (28 hpi) at 6% parasitaemia and 2% hematocrit. Infected RBCs (2x108) were treated with 10x the EC50 of compound 3 for 1 h, after which metabolites were extracted. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.
Sample Type:Blood (whole)

Treatment:

Treatment ID:TR002205
Treatment Summary:Pf3D7 cultures underwent double sorbitol synchronization 14 h apart, followed by further incubation for 28-42 h to achieve the desired trophozoite stage (28 hpi) at 6% parasitaemia and 2% hematocrit. Infected RBCs (2x108) were treated with 10x the EC50 of compound 3 for 1 h, after which metabolites were extracted. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Sample Preparation:

Sampleprep ID:SP002199
Sampleprep Summary:Pf3D7 cultures underwent double sorbitol synchronization 14 h apart, followed by further incubation for 28-42 h to achieve the desired trophozoite stage (28 hpi) at 6% parasitaemia and 2% hematocrit. Infected RBCs (2x108) were treated with 10x the EC50 of compound 3 for 1 h, after which metabolites were extracted. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2. All samples were centrifuged at 650 g for 3 min, the supernatant was removed, and the pellet washed in 500 µL of ice-cold PBS. Samples were again centrifuged at 650 g for 3 min and pellets were resuspended in 150 µL of ice-cold extraction buffer (100% methanol) and quickly resuspended. The samples were then incubated on a vortex mixer for 1 h at 4°C before being centrifuged at 17,000 g for 10 min; from this 100 µL of supernatant was collected and stored at -80°C until analysis. For each sample, another 10 µL was collected and pooled, to serve as a quality control (QC) sample.

Combined analysis:

Analysis ID AN003448 AN003449
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck) ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,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 relative intensity relative intensity

Chromatography:

Chromatography ID:CH002547
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck)
Chromatography Type:HILIC

MS:

MS ID:MS003211
Analysis ID:AN003448
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:MS003212
Analysis ID:AN003449
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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