#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