Summary of Study ST002792

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 PR001740. The data can be accessed directly via it's Project DOI: 10.21228/M8SQ7X 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	ST002792
Study Title	Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria
Study Summary	All current treatments for malaria are threatened by drug resistance, and new drug candidates that act on novel pathways are urgently needed. Here, we describe MIPS2673, a selective inhibitor of the Plasmodium M1 alanyl metalloaminopeptidase, which displays excellent in vitro antimalarial activity with no significant host cell toxicity. Biochemical assays revealed potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (Pv-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases. Orthogonal chemoproteomic methods based on thermal stability and limited proteolysis reproducibly identified PfA-M1 as the sole target of MIPS2673 in parasites from approximately 2,000 detected proteins. Furthermore, the limited proteolysis approach enabled estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Functional investigation by untargeted metabolomics further demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our proteomics and metabolomics target deconvolution strategies provided unbiased confirmation of the on-target activity of a PfA-M1 inhibitor, and validated selective inhibition of this enzyme as a promising multi-stage and cross-species antimalarial strategy.
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	2023-07-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-08-10
Release Version	1

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Project:

Project ID:	PR001740
Project DOI:	doi: 10.21228/M8SQ7X
Project Title:	Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria
Project Summary:	All current treatments for malaria are threatened by drug resistance, and new drug candidates that act on novel pathways are urgently needed. Here, we describe MIPS2673, a selective inhibitor of the Plasmodium M1 alanyl metalloaminopeptidase, which displays excellent in vitro antimalarial activity with no significant host cell toxicity. Biochemical assays revealed potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (Pv-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases. Orthogonal chemoproteomic methods based on thermal stability and limited proteolysis reproducibly identified PfA-M1 as the sole target of MIPS2673 in parasites from approximately 2,000 detected proteins. Furthermore, the limited proteolysis approach enabled estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Functional investigation by untargeted metabolomics further demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our proteomics and metabolomics target deconvolution strategies provided unbiased confirmation of the on-target activity of a PfA-M1 inhibitor, and validated selective inhibition of this enzyme as a promising multi-stage and cross-species antimalarial strategy.
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:	SU002899
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	treatment
SA299546	DMSO_iRBC_p_6	DMSO
SA299547	DMSO_iRBC_p_5	DMSO
SA299548	DMSO_iRBC_p_7	DMSO
SA299549	DMSO_iRBC_p_8	DMSO
SA299550	DMSO_iRBC_p_9	DMSO
SA299551	DMSO_iRBC_p_4	DMSO
SA299552	DMSO_iRBC_p_3	DMSO
SA299553	DMSO_iRBC_p_2	DMSO
SA299554	DMSO_iRBC_p_1	DMSO
SA299555	73_irbcs_p_2	MIPS2673
SA299556	73_irbcs_p_3	MIPS2673
SA299557	73_irbcs_p_4	MIPS2673
SA299558	73_irbcs_p_1	MIPS2673

Showing results 1 to 13 of 13

Showing results 1 to 13 of 13

Collection:

Collection ID:	CO002892
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 MIPS2673 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:	TR002908
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 MIPS2673 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:

TR002908

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 MIPS2673 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:	SP002905
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 MIPS2673 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:

SP002905

Sampleprep Summary:

Chromatography:

Chromatography ID:	CH003412
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck)
Column Temperature:	25
Flow Gradient:	0% B to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min.
Flow Rate:	0.3 ml/min
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN004542
Analysis Type:	MS
Chromatography ID:	CH003412
Num Factors:	2
Num Metabolites:	503
Rt Units:	Minutes
Units:	peak height

Analysis ID:	AN004543
Analysis Type:	MS
Chromatography ID:	CH003412
Num Factors:	2
Num Metabolites:	351
Rt Units:	Minutes
Units:	peak height