{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001238","ANALYSIS_ID":"AN002056","VERSION":"1","CREATED_ON":"August 13, 2019, 4:25 pm"},

"PROJECT":{"PROJECT_TITLE":"Antimalarial pantothenamide metabolites target acetyl-CoA biosynthesis in Plasmodium falciparum","PROJECT_SUMMARY":"Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report the discovery of potent pantothenamide bioisosteres that are active against blood-stage Plasmodium falciparum parasites and that block transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties and cleared parasites in a humanized mouse infection model of P. falciparum. Metabolomics revealed that CoA biosynthetic enzymes converted pantothenamides into CoA-analogs that interfered with parasite acetyl-CoA anabolism. In vitro generated resistant parasites showed mutations in acetyl-CoA synthetase and acyl-CoA synthetase 11. Introduction and reversion of these mutations in P. falciparum by CRISPR/Cas9 gene editing confirmed the key roles of these enzymes in the sensitivity of the malaria parasite to pantothenamides. These pantothenamide compounds with a unique mode of action may have potential as drugs against malaria parasites.","INSTITUTE":"Penn State","LAST_NAME":"Llinas","FIRST_NAME":"Manuel","ADDRESS":"W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA","EMAIL":"mul27@psu.edu","PHONE":"(814) 867-3527"},

"STUDY":{"STUDY_TITLE":"P falciparum asexual metabolomics following drug treatment (part-I)","STUDY_SUMMARY":"P falciparum infected human red blood cells were treated with 10X IC50 drug for 2.5 hours, followed by extraction and analysis of polar metabolites using HPLC-MS or HPLC-MS/MS","INSTITUTE":"Penn State","LAST_NAME":"Llinas","FIRST_NAME":"Manuel","ADDRESS":"W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA","EMAIL":"mul27@psu.edu","PHONE":"(814) 867-3527"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Plasmodium falciparum","TAXONOMY_ID":"5833"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"ND_1",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"1"}
},
{
"Subject ID":"-",
"Sample ID":"052_1",
"Factors":{"Treatment":"CXP18.6-052"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"1"}
},
{
"Subject ID":"-",
"Sample ID":"026_1",
"Factors":{"Treatment":"CXP18.6-026"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"1"}
},
{
"Subject ID":"-",
"Sample ID":"017_1",
"Factors":{"Treatment":"CXP18.6-017"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"1"}
},
{
"Subject ID":"-",
"Sample ID":"ND_2",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"2"}
},
{
"Subject ID":"-",
"Sample ID":"052_2",
"Factors":{"Treatment":"CXP18.6-052"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"2"}
},
{
"Subject ID":"-",
"Sample ID":"026_2",
"Factors":{"Treatment":"CXP18.6-026"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"2"}
},
{
"Subject ID":"-",
"Sample ID":"017_2",
"Factors":{"Treatment":"CXP18.6-017"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"2"}
},
{
"Subject ID":"-",
"Sample ID":"ND_3",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"3"}
},
{
"Subject ID":"-",
"Sample ID":"052_3",
"Factors":{"Treatment":"CXP18.6-052"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"3"}
},
{
"Subject ID":"-",
"Sample ID":"026_3",
"Factors":{"Treatment":"CXP18.6-026"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"3"}
},
{
"Subject ID":"-",
"Sample ID":"017_3",
"Factors":{"Treatment":"CXP18.6-017"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"3"}
},
{
"Subject ID":"-",
"Sample ID":"ND_1a",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"4"}
},
{
"Subject ID":"-",
"Sample ID":"258_1",
"Factors":{"Treatment":"MMV689258"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"4"}
},
{
"Subject ID":"-",
"Sample ID":"ND_2a",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"5"}
},
{
"Subject ID":"-",
"Sample ID":"258_2",
"Factors":{"Treatment":"MMV689258"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"5"}
},
{
"Subject ID":"-",
"Sample ID":"ND_3a",
"Factors":{"Treatment":"None"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"N/A","Trial":"6"}
},
{
"Subject ID":"-",
"Sample ID":"258_3",
"Factors":{"Treatment":"MMV689258"},
"Additional sample data":{"Time (hours)":"2.5","Concentration":"10X IC50","Trial":"6"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For asexual metabolomics studies all parasites were grown in standard RPMI1640 containing ~1μM pantothenic acid and supplemented with 0.25% Albumax II (Gibco). 3D7 was cultured and magnetically purified using a MACS column.","SAMPLE_TYPE":"Plasmodium cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Briefly, trophozoite stage 3D7 parasites were magnetically purified, allowed to recover in RPMI1640 (0.25% Albumax II) at 0.4% parasitemia (1x10^8 cells/sample), and treated with drug (10X IC50) for 2.5 hours."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Following treatment parasites were pelleted, washed with 1 mL of ice-cold 1X PBS, and extracted using 1 mL of ice-cold 9:1 MeOH:Water, containing the internal standard 13C4,15N1-Aspartate. Supernatants were clarified before drying under nitrogen, followed by resuspension in HPLC grade water containing 1 µM chlorpropamide to 1x10^6 parasites/µL for HPLC-MS analysis. 10 µL was injected on a Thermo Exactive Plus Orbitrap mass spectrometer for HPLC-MS-based targeted metabolomics (modified from Lu W et al 2010)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Phenomenex Synergi Hydro RP 100 A (100 x 2mm, 2.5um)"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Data was centroided to .mzXML for analysis in mzMine and Maven"},

"MS_METABOLITE_DATA":{
"Units":"Fold change versus untreated",

"Data":[{"Metabolite":"4-phosphopantothenate","ND_1":"1","052_1":"0.859863656","026_1":"0.129736113","017_1":"0.272618521","ND_2":"1","052_2":"0.795477606","026_2":"0.113432119","017_2":"0.227967542","ND_3":"1","052_3":"0.790986456","026_3":"0.191741929","017_3":"0.324606661","ND_1a":"1","258_1":"0.803420713","ND_2a":"1","258_2":"0.853595557","ND_3a":"1","258_3":"0.83843436"},{"Metabolite":"acetyl-CoA","ND_1":"1","052_1":"0.361142547","026_1":"0.332507935","017_1":"0.244158436","ND_2":"1","052_2":"0.550024194","026_2":"0.441159796","017_2":"0.431357384","ND_3":"1","052_3":"0.740112753","026_3":"0.590059252","017_3":"0.628188904","ND_1a":"1","258_1":"0.645949339","ND_2a":"1","258_2":"0.590796887","ND_3a":"1","258_3":"0.469902428"},{"Metabolite":"coenzyme A","ND_1":"1","052_1":"0.504273042","026_1":"0.706164307","017_1":"0.525684369","ND_2":"1","052_2":"0.848423174","026_2":"0.932598051","017_2":"0.796638661","ND_3":"1","052_3":"1.555324915","026_3":"1.388009447","017_3":"1.126957992","ND_1a":"1","258_1":"0.892417767","ND_2a":"1","258_2":"0.827800551","ND_3a":"1","258_3":"0.931928347"},{"Metabolite":"pantothenate","ND_1":"1","052_1":"1.172881736","026_1":"1.153136877","017_1":"1.135761938","ND_2":"1","052_2":"1.078274377","026_2":"0.950258585","017_2":"1.00008899","ND_3":"1","052_3":"1.187045555","026_3":"1.100175871","017_3":"1.224267976","ND_1a":"1","258_1":"1.036384346","ND_2a":"1","258_2":"1.070087004","ND_3a":"1","258_3":"1.132817716"}],

"Metabolites":[{"Metabolite":"pantothenate","Retention Times":"11.31","quantitated m/z":"219.1107","PubChem ID":"988","KEGG ID":"C00864"},{"Metabolite":"4-phosphopantothenate","Retention Times":"13.94","quantitated m/z":"298.0697","PubChem ID":"16755653","KEGG ID":"C03492"},{"Metabolite":"coenzyme A","Retention Times":"15.93","quantitated m/z":"766.1079","PubChem ID":"87642","KEGG ID":"C00010"},{"Metabolite":"acetyl-CoA","Retention Times":"16.18","quantitated m/z":"808.1185","PubChem ID":"6302","KEGG ID":"C00024"}]
}

}