#METABOLOMICS WORKBENCH eallman_20190808_172952 DATATRACK_ID:1790 STUDY_ID:ST001238 ANALYSIS_ID:AN002057 PROJECT_ID:PR000829
VERSION             	1
CREATED_ON             	August 13, 2019, 4:25 pm
#PROJECT
PR:PROJECT_TITLE                 	Antimalarial pantothenamide metabolites target acetyl-CoA biosynthesis in
PR:PROJECT_TITLE                 	Plasmodium falciparum
PR:PROJECT_SUMMARY               	Malaria eradication is critically dependent on new therapeutics that target
PR:PROJECT_SUMMARY               	resistant Plasmodium parasites and block transmission of the disease. Here, we
PR:PROJECT_SUMMARY               	report the discovery of potent pantothenamide bioisosteres that are active
PR:PROJECT_SUMMARY               	against blood-stage Plasmodium falciparum parasites and that block transmission
PR:PROJECT_SUMMARY               	of sexual stages to the mosquito vector. These compounds were resistant to
PR:PROJECT_SUMMARY               	degradation by serum pantetheinases, showed favorable pharmacokinetic properties
PR:PROJECT_SUMMARY               	and cleared parasites in a humanized mouse infection model of P. falciparum.
PR:PROJECT_SUMMARY               	Metabolomics revealed that CoA biosynthetic enzymes converted pantothenamides
PR:PROJECT_SUMMARY               	into CoA-analogs that interfered with parasite acetyl-CoA anabolism. In vitro
PR:PROJECT_SUMMARY               	generated resistant parasites showed mutations in acetyl-CoA synthetase and
PR:PROJECT_SUMMARY               	acyl-CoA synthetase 11. Introduction and reversion of these mutations in P.
PR:PROJECT_SUMMARY               	falciparum by CRISPR/Cas9 gene editing confirmed the key roles of these enzymes
PR:PROJECT_SUMMARY               	in the sensitivity of the malaria parasite to pantothenamides. These
PR:PROJECT_SUMMARY               	pantothenamide compounds with a unique mode of action may have potential as
PR:PROJECT_SUMMARY               	drugs against malaria parasites.
PR:INSTITUTE                     	Penn State
PR:LAST_NAME                     	Llinas
PR:FIRST_NAME                    	Manuel
PR:ADDRESS                       	W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
PR:EMAIL                         	mul27@psu.edu
PR:PHONE                         	(814) 867-3527
#STUDY
ST:STUDY_TITLE                   	P falciparum asexual metabolomics following drug treatment (part-I)
ST:STUDY_SUMMARY                 	P falciparum infected human red blood cells were treated with 10X IC50 drug for
ST:STUDY_SUMMARY                 	2.5 hours, followed by extraction and analysis of polar metabolites using
ST:STUDY_SUMMARY                 	HPLC-MS or HPLC-MS/MS
ST:INSTITUTE                     	Penn State
ST:LAST_NAME                     	Llinas
ST:FIRST_NAME                    	Manuel
ST:ADDRESS                       	W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
ST:EMAIL                         	mul27@psu.edu
ST:PHONE                         	(814) 867-3527
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Plasmodium falciparum
SU:TAXONOMY_ID                   	5833
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	ND_1	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=1
SUBJECT_SAMPLE_FACTORS           	-	052_1	Treatment:CXP18.6-052	Time (hours)=2.5; Concentration=10X IC50; Trial=1
SUBJECT_SAMPLE_FACTORS           	-	026_1	Treatment:CXP18.6-026	Time (hours)=2.5; Concentration=10X IC50; Trial=1
SUBJECT_SAMPLE_FACTORS           	-	017_1	Treatment:CXP18.6-017	Time (hours)=2.5; Concentration=10X IC50; Trial=1
SUBJECT_SAMPLE_FACTORS           	-	ND_2	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=2
SUBJECT_SAMPLE_FACTORS           	-	052_2	Treatment:CXP18.6-052	Time (hours)=2.5; Concentration=10X IC50; Trial=2
SUBJECT_SAMPLE_FACTORS           	-	026_2	Treatment:CXP18.6-026	Time (hours)=2.5; Concentration=10X IC50; Trial=2
SUBJECT_SAMPLE_FACTORS           	-	017_2	Treatment:CXP18.6-017	Time (hours)=2.5; Concentration=10X IC50; Trial=2
SUBJECT_SAMPLE_FACTORS           	-	ND_3	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=3
SUBJECT_SAMPLE_FACTORS           	-	052_3	Treatment:CXP18.6-052	Time (hours)=2.5; Concentration=10X IC50; Trial=3
SUBJECT_SAMPLE_FACTORS           	-	026_3	Treatment:CXP18.6-026	Time (hours)=2.5; Concentration=10X IC50; Trial=3
SUBJECT_SAMPLE_FACTORS           	-	017_3	Treatment:CXP18.6-017	Time (hours)=2.5; Concentration=10X IC50; Trial=3
SUBJECT_SAMPLE_FACTORS           	-	ND_1a	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=4
SUBJECT_SAMPLE_FACTORS           	-	258_1	Treatment:MMV689258	Time (hours)=2.5; Concentration=10X IC50; Trial=4
SUBJECT_SAMPLE_FACTORS           	-	ND_2a	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=5
SUBJECT_SAMPLE_FACTORS           	-	258_2	Treatment:MMV689258	Time (hours)=2.5; Concentration=10X IC50; Trial=5
SUBJECT_SAMPLE_FACTORS           	-	ND_3a	Treatment:None	Time (hours)=2.5; Concentration=N/A; Trial=6
SUBJECT_SAMPLE_FACTORS           	-	258_3	Treatment:MMV689258	Time (hours)=2.5; Concentration=10X IC50; Trial=6
#COLLECTION
CO:COLLECTION_SUMMARY            	For asexual metabolomics studies all parasites were grown in standard RPMI1640
CO:COLLECTION_SUMMARY            	containing ~1μM pantothenic acid and supplemented with 0.25% Albumax II
CO:COLLECTION_SUMMARY            	(Gibco). 3D7 was cultured and magnetically purified using a MACS column.
CO:SAMPLE_TYPE                   	Plasmodium cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Briefly, trophozoite stage 3D7 parasites were magnetically purified, allowed to
TR:TREATMENT_SUMMARY             	recover in RPMI1640 (0.25% Albumax II) at 0.4% parasitemia (1x10^8
TR:TREATMENT_SUMMARY             	cells/sample), and treated with drug (10X IC50) for 2.5 hours.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Following treatment parasites were pelleted, washed with 1 mL of ice-cold 1X
SP:SAMPLEPREP_SUMMARY            	PBS, and extracted using 1 mL of ice-cold 9:1 MeOH:Water, containing the
SP:SAMPLEPREP_SUMMARY            	internal standard 13C4,15N1-Aspartate. Supernatants were clarified before drying
SP:SAMPLEPREP_SUMMARY            	under nitrogen, followed by resuspension in HPLC grade water containing 1 µM
SP:SAMPLEPREP_SUMMARY            	chlorpropamide to 1x10^6 parasites/µL for HPLC-MS analysis. 10 µL was injected
SP:SAMPLEPREP_SUMMARY            	on a Thermo Exactive Plus Orbitrap mass spectrometer for HPLC-MS-based targeted
SP:SAMPLEPREP_SUMMARY            	metabolomics (modified from Lu W et al 2010).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Shimadzu Prominence 20 UFLCXR
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (100 x 2mm, 1.7um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 5600 TripleTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Data was centroided to .mzXML for analysis in mzMine and Maven
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Intensity
MS_METABOLITE_DATA_START
Samples	052_1	017_1	258_1	026_1
Factors	Treatment:CXP18.6-052	Treatment:CXP18.6-017	Treatment:MMV689258	Treatment:CXP18.6-026
CXP18.6-052	870.7874	0	0	0
CXP18.6-017	0	10990.1191	0	0
MMV689258	0	0	7756.9175	0
CXP18.6-026	0	0	0	4194.5225
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention Time	quantitated m/z	PubChem ID	KEGG ID
CXP18.6-052	2.65	322.1777
CXP18.6-017	7.74	339.1729
MMV689258	8.47	353.1882
CXP18.6-026	8.59	335.1978
METABOLITES_END
#END