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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Study ID	ST002108
Study Title	Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway (Part 3)
Study 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
Submit Date	2022-03-17
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-04-04
Release Version	1

Select appropriate tab below to view additional metadata details:

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
Species Group:	Unicellular parasites

Factors:

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

mb_sample_id	local_sample_id	cell_type	treatment
SA202348	71_irbcs_p_1	iRBC	Compound 3
SA202349	71_irbcs_p_2	iRBC	Compound 3
SA202350	71_irbcs_p_4	iRBC	Compound 3
SA202351	71_irbcs_p_3	iRBC	Compound 3
SA202352	DMSO_iRBC_p_7	iRBC	DMSO
SA202353	DMSO_iRBC_p_8	iRBC	DMSO
SA202354	DMSO_iRBC_p_6	iRBC	DMSO
SA202355	DMSO_iRBC_p_9	iRBC	DMSO
SA202356	DMSO_iRBC_p_3	iRBC	DMSO
SA202357	DMSO_iRBC_p_1	iRBC	DMSO
SA202358	DMSO_iRBC_p_2	iRBC	DMSO
SA202359	DMSO_iRBC_p_4	iRBC	DMSO
SA202360	DMSO_iRBC_p_5	iRBC	DMSO

Showing results 1 to 13 of 13

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.

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.

Sampleprep ID:

SP002199

Sampleprep Summary:

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

Analysis:

Analysis ID:	AN003448
Analysis Type:	MS
Chromatography ID:	CH002547
Num Factors:	2
Num Metabolites:	558
Rt Units:	Minutes
Units:	relative intensity

Analysis ID:	AN003449
Analysis Type:	MS
Chromatography ID:	CH002547
Num Factors:	2
Num Metabolites:	439
Rt Units:	Minutes
Units:	relative intensity