{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002792","ANALYSIS_ID":"AN004542","VERSION":"1","CREATED_ON":"July 24, 2023, 8:29 am"},

"PROJECT":{"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"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Plasmodium falciparum","TAXONOMY_ID":"5833"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"73_irbcs_p_1",
"Factors":{"treatment":"MIPS2673"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"73_irbcs_p_1"}
},
{
"Subject ID":"-",
"Sample ID":"73_irbcs_p_2",
"Factors":{"treatment":"MIPS2673"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"73_irbcs_p_2"}
},
{
"Subject ID":"-",
"Sample ID":"73_irbcs_p_3",
"Factors":{"treatment":"MIPS2673"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"73_irbcs_p_3"}
},
{
"Subject ID":"-",
"Sample ID":"73_irbcs_p_4",
"Factors":{"treatment":"MIPS2673"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"73_irbcs_p_4"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_1",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_1"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_2",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_2"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_3",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_4",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_4"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_5",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_6",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_7",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_7"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_8",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","RAW_FILE_NAME":"DMSO_iRBC_p_8"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_iRBC_p_9",
"Factors":{"treatment":"DMSO"},
"Additional sample data":{"cell_type":"iRBC","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 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_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":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"ZIC-pHILIC (150 x 4.6mm,5um) equipped with a guard (SeQuant,Merck)","SOLVENT_A":"100% water; 20 mM ammonium carbonate","SOLVENT_B":"100% acetonitrile","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","COLUMN_TEMPERATURE":"25"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","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":"peak height",

"Data":[{"Metabolite":"1H-Indole-3-acetamide","73_irbcs_p_1":"238585","73_irbcs_p_2":"217392","73_irbcs_p_3":"828408","73_irbcs_p_4":"463868","DMSO_iRBC_p_1":"630816","DMSO_iRBC_p_2":"100915","DMSO_iRBC_p_3":"730411","DMSO_iRBC_p_4":"1167192","DMSO_iRBC_p_5":"636979","DMSO_iRBC_p_6":"478586","DMSO_iRBC_p_7":"129992","DMSO_iRBC_p_8":"175138","DMSO_iRBC_p_9":"510625"},{"Metabolite":"Chorismate","73_irbcs_p_1":"152738","73_irbcs_p_2":"185879","73_irbcs_p_3":"258302","73_irbcs_p_4":"215404","DMSO_iRBC_p_1":"143317","DMSO_iRBC_p_2":"97611","DMSO_iRBC_p_3":"186679","DMSO_iRBC_p_4":"161176","DMSO_iRBC_p_5":"109768","DMSO_iRBC_p_6":"127123","DMSO_iRBC_p_7":"138612","DMSO_iRBC_p_8":"128728","DMSO_iRBC_p_9":"162874"},{"Metabolite":"Ferulic acid 4-sulfate","73_irbcs_p_1":"0","73_irbcs_p_2":"68449","73_irbcs_p_3":"172691","73_irbcs_p_4":"81847","DMSO_iRBC_p_1":"191649","DMSO_iRBC_p_2":"57168","DMSO_iRBC_p_3":"124446","DMSO_iRBC_p_4":"122412","DMSO_iRBC_p_5":"214414","DMSO_iRBC_p_6":"232330","DMSO_iRBC_p_7":"0","DMSO_iRBC_p_8":"227844","DMSO_iRBC_p_9":"67939"},{"Metabolite":"Kynurenine","73_irbcs_p_1":"0","73_irbcs_p_2":"107444","73_irbcs_p_3":"107026","73_irbcs_p_4":"0","DMSO_iRBC_p_1":"37673","DMSO_iRBC_p_2":"144072","DMSO_iRBC_p_3":"88547","DMSO_iRBC_p_4":"199518","DMSO_iRBC_p_5":"279786","DMSO_iRBC_p_6":"108821","DMSO_iRBC_p_7":"0","DMSO_iRBC_p_8":"85763","DMSO_iRBC_p_9":"0"},{"Metabolite":"Inosine","73_irbcs_p_1":"216143","73_irbcs_p_2":"157644","73_irbcs_p_3":"659908","73_irbcs_p_4":"364014","DMSO_iRBC_p_1":"324032","DMSO_iRBC_p_2":"316408","DMSO_iRBC_p_3":"1337869","DMSO_iRBC_p_4":"599161","DMSO_iRBC_p_5":"878094","DMSO_iRBC_p_6":"1008027","DMSO_iRBC_p_7":"239477","DMSO_iRBC_p_8":"173122","DMSO_iRBC_p_9":"327287"},{"Metabolite":"thioacetazone","73_irbcs_p_1":"63655","73_irbcs_p_2":"63139","73_irbcs_p_3":"71654","73_irbcs_p_4":"68980","DMSO_iRBC_p_1":"73165","DMSO_iRBC_p_2":"50998","DMSO_iRBC_p_3":"32636","DMSO_iRBC_p_4":"38556","DMSO_iRBC_p_5":"33518","DMSO_iRBC_p_6":"22350","DMSO_iRBC_p_7":"42831","DMSO_iRBC_p_8":"43827","DMSO_iRBC_p_9":"40708"},{"Metabolite":"2-(4-sulfophenyl)butyrate","73_irbcs_p_1":"48198","73_irbcs_p_2":"75132","73_irbcs_p_3":"57571","73_irbcs_p_4":"63424","DMSO_iRBC_p_1":"88191","DMSO_iRBC_p_2":"97739","DMSO_iRBC_p_3":"62299","DMSO_iRBC_p_4":"0","DMSO_iRBC_p_5":"27090","DMSO_iRBC_p_6":"0","DMSO_iRBC_p_7":"30283","DMSO_iRBC_p_8":"33770","DMSO_iRBC_p_9":"25097"},{"Metabolite":"dIMP","73_irbcs_p_1":"1122398","73_irbcs_p_2":"812270","73_irbcs_p_3":"541262","73_irbcs_p_4":"698227","DMSO_iRBC_p_1":"905074","DMSO_iRBC_p_2":"515004","DMSO_iRBC_p_3":"1768678","DMSO_iRBC_p_4":"1065166","DMSO_iRBC_p_5":"1277072","DMSO_iRBC_p_6":"1481692","DMSO_iRBC_p_7":"1207687","DMSO_iRBC_p_8":"985754","DMSO_iRBC_p_9":"1131649"},{"Metabolite":"6-Hydroxyl-1,6-dihydropurine ribonucleoside","73_irbcs_p_1":"233365","73_irbcs_p_2":"236746","73_irbcs_p_3":"310699","73_irbcs_p_4":"210757","DMSO_iRBC_p_1":"111397","DMSO_iRBC_p_2":"122926","DMSO_iRBC_p_3":"226943","DMSO_iRBC_p_4":"284231","DMSO_iRBC_p_5":"305728","DMSO_iRBC_p_6":"310128","DMSO_iRBC_p_7":"261838","DMSO_iRBC_p_8":"225335","DMSO_iRBC_p_9":"188217"},{"Metabolite":"Adenosine monophosphate","73_irbcs_p_1":"3364455","73_irbcs_p_2":"4665005","73_irbcs_p_3":"5557097","73_irbcs_p_4":"3696445","DMSO_iRBC_p_1":"2550342","DMSO_iRBC_p_2":"4550285","DMSO_iRBC_p_3":"10595683","DMSO_iRBC_p_4":"8248633","DMSO_iRBC_p_5":"18101794","DMSO_iRBC_p_6":"7897584","DMSO_iRBC_p_7":"4059205","DMSO_iRBC_p_8":"4545717","DMSO_iRBC_p_9":"3740269"},{"Metabolite":"Asn/His","73_irbcs_p_1":"138128","73_irbcs_p_2":"134168","73_irbcs_p_3":"145425","73_irbcs_p_4":"140267","DMSO_iRBC_p_1":"44695","DMSO_iRBC_p_2":"35453","DMSO_iRBC_p_3":"14596","DMSO_iRBC_p_4":"20534","DMSO_iRBC_p_5":"50454","DMSO_iRBC_p_6":"67880","DMSO_iRBC_p_7":"42187","DMSO_iRBC_p_8":"42206","DMSO_iRBC_p_9":"22525"},{"Metabolite":"2-(4-Methyl-5-thiazolyl)ethyl butanoate","73_irbcs_p_1":"155002","73_irbcs_p_2":"132604","73_irbcs_p_3":"114961","73_irbcs_p_4":"145933","DMSO_iRBC_p_1":"56646","DMSO_iRBC_p_2":"70436","DMSO_iRBC_p_3":"105128","DMSO_iRBC_p_4":"110509","DMSO_iRBC_p_5":"70819","DMSO_iRBC_p_6":"61363","DMSO_iRBC_p_7":"63200","DMSO_iRBC_p_8":"63763","DMSO_iRBC_p_9":"66639"},{"Metabolite":"Biotin","73_irbcs_p_1":"35343","73_irbcs_p_2":"43187","73_irbcs_p_3":"48831","73_irbcs_p_4":"33230","DMSO_iRBC_p_1":"38803","DMSO_iRBC_p_2":"30094","DMSO_iRBC_p_3":"42977","DMSO_iRBC_p_4":"77113","DMSO_iRBC_p_5":"81358","DMSO_iRBC_p_6":"44455","DMSO_iRBC_p_7":"42967","DMSO_iRBC_p_8":"31208","DMSO_iRBC_p_9":"35693"},{"Metabolite":"Thr/His","73_irbcs_p_1":"53783","73_irbcs_p_2":"55262","73_irbcs_p_3":"36612","73_irbcs_p_4":"52944","DMSO_iRBC_p_1":"0","DMSO_iRBC_p_2":"0","DMSO_iRBC_p_3":"0","DMSO_iRBC_p_4":"0","DMSO_iRBC_p_5":"0","DMSO_iRBC_p_6":"0","DMSO_iRBC_p_7":"0","DMSO_iRBC_p_8":"0","DMSO_iRBC_p_9":"0"},{"Metabolite":"1-O-malyl-beta-D-glucose","73_irbcs_p_1":"0","73_irbcs_p_2":"0","73_irbcs_p_3":"453941","73_irbcs_p_4":"51609","DMSO_iRBC_p_1":"415980","DMSO_iRBC_p_2":"51619","DMSO_iRBC_p_3":"574754","DMSO_iRBC_p_4":"369744","DMSO_iRBC_p_5":"750684","DMSO_iRBC_p_6":"873000","DMSO_iRBC_p_7":"0","DMSO_iRBC_p_8":"317986","DMSO_iRBC_p_9":"88198"},{"Metabolite":"Biotin 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}

}