{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002108","ANALYSIS_ID":"AN003449","VERSION":"1","CREATED_ON":"March 18, 2022, 8:02 am"},

"PROJECT":{"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"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Plasmodium falciparum","TAXONOMY_ID":"5833"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"71_irbcs_p_1",
"Factors":{"cell_type":"iRBC","treatment":"Compound 3"},
"Additional sample data":{"RAW_FILE_NAME":"71_irbcs_p_1"}
},
{
"Subject ID":"-",
"Sample ID":"71_irbcs_p_2",
"Factors":{"cell_type":"iRBC","treatment":"Compound 3"},
"Additional sample data":{"RAW_FILE_NAME":"71_irbcs_p_2"}
},
{
"Subject ID":"-",
"Sample ID":"71_irbcs_p_3",
"Factors":{"cell_type":"iRBC","treatment":"Compound 3"},
"Additional sample data":{"RAW_FILE_NAME":"71_irbcs_p_3"}
},
{
"Subject ID":"-",
"Sample ID":"71_irbcs_p_4",
"Factors":{"cell_type":"iRBC","treatment":"Compound 3"},
"Additional sample data":{"RAW_FILE_NAME":"71_irbcs_p_4"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_1",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_1"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_2",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_2"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_3",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_4",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_4"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_5",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_6",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_7",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_7"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_8",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_8"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_9",
"Factors":{"cell_type":"iRBC","treatment":"DMSO"},
"Additional sample data":{"RAW_FILE_NAME":"DMSO_iRBC_p_9"}
}
],
"COLLECTION":{"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_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":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"ZIC-pHILIC column equipped with a guard (5 μm, 4.6 × 150 mm, SeQuant®, Merck)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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."},

"MS_METABOLITE_DATA":{
"Units":"relative intensity",

"Data":[{"Metabolite":"Malonic semialdehyde","71_irbcs_p_1":"206566.5781","71_irbcs_p_2":"287964.1563","71_irbcs_p_3":"302496.875","71_irbcs_p_4":"194619.2969","DMSO_iRBC_p_1":"248287.9844","DMSO_iRBC_p_2":"323221.9688","DMSO_iRBC_p_3":"277052.5938","DMSO_iRBC_p_4":"420295.0313","DMSO_iRBC_p_5":"371413.6875","DMSO_iRBC_p_6":"365375.3438","DMSO_iRBC_p_7":"220826.9219","DMSO_iRBC_p_8":"280007","DMSO_iRBC_p_9":"189339.8438"},{"Metabolite":"3-Hydroxypropenoate","71_irbcs_p_1":"54246.61719","71_irbcs_p_2":"119660.8438","71_irbcs_p_3":"170041.6094","71_irbcs_p_4":"102989.5938","DMSO_iRBC_p_1":"104912.4219","DMSO_iRBC_p_2":"148859.6094","DMSO_iRBC_p_3":"168086.625","DMSO_iRBC_p_4":"295166.9375","DMSO_iRBC_p_5":"308328.3438","DMSO_iRBC_p_6":"223737.4375","DMSO_iRBC_p_7":"59462.70703","DMSO_iRBC_p_8":"105423.25","DMSO_iRBC_p_9":"87589.52344"},{"Metabolite":"Pyruvic acid","71_irbcs_p_1":"47351.03125","71_irbcs_p_2":"95691.67188","71_irbcs_p_3":"87032.28906","71_irbcs_p_4":"25474.88086","DMSO_iRBC_p_1":"41452.38281","DMSO_iRBC_p_2":"81006.60938","DMSO_iRBC_p_3":"42562.03125","DMSO_iRBC_p_4":"177628.9688","DMSO_iRBC_p_5":"63258.05078","DMSO_iRBC_p_6":"110028.7188","DMSO_iRBC_p_7":"66122.14063","DMSO_iRBC_p_8":"101774.6797","DMSO_iRBC_p_9":"41311.98438"},{"Metabolite":"Lactic acid","71_irbcs_p_1":"3969823","71_irbcs_p_2":"2672750.25","71_irbcs_p_3":"6995493.5","71_irbcs_p_4":"1436307.25","DMSO_iRBC_p_1":"1782547.625","DMSO_iRBC_p_2":"3362834.5","DMSO_iRBC_p_3":"2316663","DMSO_iRBC_p_4":"7545603.5","DMSO_iRBC_p_5":"6698687.5","DMSO_iRBC_p_6":"8493017","DMSO_iRBC_p_7":"6216986.5","DMSO_iRBC_p_8":"2881176.25","DMSO_iRBC_p_9":"1709005.75"},{"Metabolite":"Phosphoramidate","71_irbcs_p_1":"321259.6875","71_irbcs_p_2":"399564.5313","71_irbcs_p_3":"544361.1875","71_irbcs_p_4":"454657.8125","DMSO_iRBC_p_1":"373561.8125","DMSO_iRBC_p_2":"421461.5313","DMSO_iRBC_p_3":"441965.5938","DMSO_iRBC_p_4":"521737.4375","DMSO_iRBC_p_5":"486955.2188","DMSO_iRBC_p_6":"452300.8438","DMSO_iRBC_p_7":"420973.1875","DMSO_iRBC_p_8":"400489.4375","DMSO_iRBC_p_9":"407139.5313"},{"Metabolite":"Hydrogen phosphate","71_irbcs_p_1":"13400000","71_irbcs_p_2":"14700000","71_irbcs_p_3":"17700000","71_irbcs_p_4":"13900000","DMSO_iRBC_p_1":"13700000","DMSO_iRBC_p_2":"15200000","DMSO_iRBC_p_3":"19200000","DMSO_iRBC_p_4":"21800000","DMSO_iRBC_p_5":"19500000","DMSO_iRBC_p_6":"16700000","DMSO_iRBC_p_7":"16000000","DMSO_iRBC_p_8":"15300000","DMSO_iRBC_p_9":"14400000"},{"Metabolite":"Hexanal","71_irbcs_p_1":"58943.82422","71_irbcs_p_2":"231798.2344","71_irbcs_p_3":"238148.1563","71_irbcs_p_4":"167084.7344","DMSO_iRBC_p_1":"186570.6563","DMSO_iRBC_p_2":"128408.1953","DMSO_iRBC_p_3":"100885.9922","DMSO_iRBC_p_4":"185365.8281","DMSO_iRBC_p_5":"211571.125","DMSO_iRBC_p_6":"130621.6875","DMSO_iRBC_p_7":"91130.96094","DMSO_iRBC_p_8":"218274.375","DMSO_iRBC_p_9":"119794.875"},{"Metabolite":"Acetoacetic acid","71_irbcs_p_1":"95879.47656","71_irbcs_p_2":"352951.9375","71_irbcs_p_3":"520167.2813","71_irbcs_p_4":"365371.9375","DMSO_iRBC_p_1":"134669.8906","DMSO_iRBC_p_2":"287948.9063","DMSO_iRBC_p_3":"367636.4063","DMSO_iRBC_p_4":"414148.4688","DMSO_iRBC_p_5":"434913.7813","DMSO_iRBC_p_6":"307721.1563","DMSO_iRBC_p_7":"113439.2422","DMSO_iRBC_p_8":"313047.8125","DMSO_iRBC_p_9":"332333.7813"},{"Metabolite":"Hydroxypyruvic acid","71_irbcs_p_1":"0","71_irbcs_p_2":"26218.08008","71_irbcs_p_3":"40267.08984","71_irbcs_p_4":"26948.84375","DMSO_iRBC_p_1":"20170.5957","DMSO_iRBC_p_2":"28333.37891","DMSO_iRBC_p_3":"53039.23438","DMSO_iRBC_p_4":"183249.4531","DMSO_iRBC_p_5":"276683.5313","DMSO_iRBC_p_6":"158458.1406","DMSO_iRBC_p_7":"46597.94922","DMSO_iRBC_p_8":"48801.30078","DMSO_iRBC_p_9":"17021.55078"},{"Metabolite":"Glyceric acid","71_irbcs_p_1":"63618.68359","71_irbcs_p_2":"72443.98438","71_irbcs_p_3":"99223.6875","71_irbcs_p_4":"57328.90625","DMSO_iRBC_p_1":"62337.19141","DMSO_iRBC_p_2":"97797.13281","DMSO_iRBC_p_3":"87372.75781","DMSO_iRBC_p_4":"84196.07813","DMSO_iRBC_p_5":"108980.4688","DMSO_iRBC_p_6":"158762.875","DMSO_iRBC_p_7":"85270.17188","DMSO_iRBC_p_8":"70901.35938","DMSO_iRBC_p_9":"48631.39453"},{"Metabolite":"Imidazole-4-acetaldehyde","71_irbcs_p_1":"78270.96875","71_irbcs_p_2":"82402.14844","71_irbcs_p_3":"71852.46875","71_irbcs_p_4":"48421.39844","DMSO_iRBC_p_1":"57610.14844","DMSO_iRBC_p_2":"64241.65625","DMSO_iRBC_p_3":"63120.29688","DMSO_iRBC_p_4":"68736.07031","DMSO_iRBC_p_5":"51712.24609","DMSO_iRBC_p_6":"74209.03125","DMSO_iRBC_p_7":"98401.125","DMSO_iRBC_p_8":"102752.6797","DMSO_iRBC_p_9":"46182.9375"},{"Metabolite":"selenium 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}

}