Summary of Study ST003565
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002199. The data can be accessed directly via it's Project DOI: 10.21228/M8CN7H This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003565 |
| Study Title | Metaboloomics analysis of the antimalarial compound WEHI-1888504 (aka compound 59) in Plasmodium falciparum (3D7) infected red blood cells |
| Study Summary | Emerging resistance to frontline antimalarials is resulting in them becoming increasingly ineffective, highlighting the need for new antimalarials. To discover new antimalarials a screen of the Janssen Jumpstarter library against the asexual stage parasite uncovered an N-acetamide indole hit class. The structure-activity relationship of this chemotype was defined by generating optimized frontrunner analogs including WJM664 with potent asexual stage activity and high metabolic stability. To determine the mechanism of action, resistance was selected and whole genome sequencing revealed mutations in PfATP4. PfATP4 was validated as the target of the N-acetamide indole class showing cross-resistance to PfATP4 drug-resistant strains, and a metabolomic signature consistent with the PfATP4 inhibitor KAE609. The indole acetamide class exhibited a fast-to-moderate rate of kill and a vacuolized asexual blood stage phenotype, phenocopying known PfATP4 inhibitors. Moreover, N-acetamide indole analogs increased cytosolic Na+ demonstrating on-target inhibition of PfATP4. N-Acetamide indole derivatives inhibited male and female gamete development and blocked transmission from infected blood to the mosquito. WJM664 exhibited low oral efficacy in an asexual stage P. berghei mouse model which may be attributed to the low potency against PbATP4, and the low aqueous solubility and Caco-2 permeability. Further optimization of these attributes is required for the N-acetamide indole class to be considered for development in a curative or transmission blocking combination therapy. |
| Institute | Monash University |
| Last Name | Giannangelo |
| First Name | Carlo |
| Address | 381 Royal Parade, Parkville, Victoria, 3052, Australia |
| carlo.giannangelo@monash.edu | |
| Phone | 99039282 |
| Submit Date | 2024-11-05 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-12-02 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002199 |
| Project DOI: | doi: 10.21228/M8CN7H |
| Project Title: | Optimization and characterization of N-acetamide indoles as antimalarials that target the Plasmodium falciparum protein, PfATP4 |
| Project Summary: | Emerging resistance to frontline antimalarials is resulting in them becoming increasingly ineffective, highlighting the need for new antimalarials. To discover new antimalarials a screen of the Janssen Jumpstarter library against the asexual stage parasite uncovered an N-acetamide indole hit class. The structure-activity relationship of this chemotype was defined by generating optimized frontrunner analogs including WJM664 with potent asexual stage activity and high metabolic stability. To determine the mechanism of action, resistance was selected and whole genome sequencing revealed mutations in PfATP4. PfATP4 was validated as the target of the N-acetamide indole class showing cross-resistance to PfATP4 drug-resistant strains, and a metabolomic signature consistent with the PfATP4 inhibitor KAE609. The indole acetamide class exhibited a fast-to-moderate rate of kill and a vacuolized asexual blood stage phenotype, phenocopying known PfATP4 inhibitors. Moreover, N-acetamide indole analogs increased cytosolic Na+ demonstrating on-target inhibition of PfATP4. N-Acetamide indole derivatives inhibited male and female gamete development and blocked transmission from infected blood to the mosquito. WJM664 exhibited low oral efficacy in an asexual stage P. berghei mouse model which may be attributed to the low potency against PbATP4, and the low aqueous solubility and Caco-2 permeability. Further optimization of these attributes is required for the N-acetamide indole class to be considered for development in a curative or transmission blocking combination therapy. |
| Institute: | Monash University |
| Last Name: | Giannangelo |
| First Name: | Carlo |
| Address: | 381 Royal Parade, Parkville, Victoria, 3052, Australia |
| Email: | carlo.giannangelo@monash.edu |
| Phone: | 99039282 |
Subject:
| Subject ID: | SU003694 |
| Subject Type: | Cultured cells |
| Subject Species: | Plasmodium falciparum |
| Taxonomy ID: | 5833 |
| Species Group: | Unicellular parasites |
Factors:
Subject type: Cultured cells; Subject species: Plasmodium falciparum (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA389455 | DMSO_1 | Pf3D7_infected_RBC | DMSO |
| SA389456 | DMSO_2 | Pf3D7_infected_RBC | DMSO |
| SA389457 | DMSO_3 | Pf3D7_infected_RBC | DMSO |
| SA389458 | DMSO_4 | Pf3D7_infected_RBC | DMSO |
| SA389459 | KAE609_20x_1 | Pf3D7_infected_RBC | KAE609_20x_IC50 |
| SA389460 | KAE609_20x_2 | Pf3D7_infected_RBC | KAE609_20x_IC50 |
| SA389461 | KAE609_20x_3 | Pf3D7_infected_RBC | KAE609_20x_IC50 |
| SA389462 | KAE609_5x_1 | Pf3D7_infected_RBC | KAE609_5x_IC50 |
| SA389463 | KAE609_5x_2 | Pf3D7_infected_RBC | KAE609_5x_IC50 |
| SA389464 | KAE609_5x_3 | Pf3D7_infected_RBC | KAE609_5x_IC50 |
| SA389465 | WEHI-1888504_1 | Pf3D7_infected_RBC | WEHI-1888504 |
| SA389466 | WEHI-1888504_2 | Pf3D7_infected_RBC | WEHI-1888504 |
| SA389467 | WEHI-1888504_3 | Pf3D7_infected_RBC | WEHI-1888504 |
| Showing results 1 to 13 of 13 |
Collection:
| Collection ID: | CO003687 |
| Collection Summary: | P. falciparum infected red blood cell (RBC) cultures (3D7 strain) synchronized to the mid trophozoite stage were magnet purified to achieve a parasitemia of >90% and hematocrit of 0.5%. After a period of recovery the culture medium was refreshed immediately before incubation with the test compounds. |
| Sample Type: | Plasmodium infected red blood cells |
Treatment:
| Treatment ID: | TR003703 |
| Treatment Summary: | The magnetically purified infected RBC cultures were incubated with 1.1 µM of the test compound WEHI-1888504 (aka analog 59, 5 × IC50), 5 nM (5 × IC50), or 20 nM (20 × IC50) of the known PfATP4 inhibitor KAE609 (1), or an equivalent volume of vehicle (DMSO). Incubation duration was for 5 h at 37C. (IC50 = 50% inhibitory concentration) |
Sample Preparation:
| Sampleprep ID: | SP003701 |
| Sampleprep Summary: | Following compound incubation, the cultures were centrifuged, the cell pellets were washed with ice-cold PBS, and the metabolites from 5 x 10^7 cells were extracted using 90 µL of ice-cold methanol. Samples were vortexed for 1 h, centrifuged and then supernatants were transferred into high-performance liquid chromatography (HPLC) vials for storage at -80 °C until liquid chromatography-mass spectrometry (LC-MS) analysis. A 10 µL aliquot from each sample was pooled to serve as a quality control sample for monitoring instrument reproducibility and to aid downstream metabolite identification. |
Combined analysis:
| Analysis ID | AN005857 | AN005858 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Orbitrap Exploris 120 | Thermo Orbitrap Exploris 120 |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Peak height | Peak height |
Chromatography:
| Chromatography ID: | CH004449 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Temperature: | 25 |
| Flow Gradient: | 0–10 min, 80–50% B; 10–12 min, 50–5% B; 12–14 min, 5% B; 14–16 min, 5–80% B and 16–22 min, 80% B |
| Flow Rate: | 0.35 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005577 |
| Analysis ID: | AN005857 |
| Instrument Name: | Thermo Orbitrap Exploris 120 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data were acquired as a full scan in positive and negative ionization modes with a heated electrospray source and an Orbitrap resolution of 120,000 from 70 to 1,050 m/z. Ion source voltage was 3,500 V in positive mode and 2,500 V in negative mode. The ion transfer tube temperature was 325 °C and the vaporizer temperature was 350 °C. Gas mode was set to static with sheath gas, aux gas, and sweep gas at 50, 10, and 1, respectively. Samples within the LC-MS batch were sorted according to blocks of replicates and randomized. To facilitate metabolite identification, approximately 350 authentic metabolite standards were analyzed before the LC-MS batch, and their peaks and retention time were manually checked using the MZmine software. Pooled biological quality control samples and extraction solvent blanks were analyzed periodically throughout the batch to monitor LC-MS signal reproducibility and assist metabolite identification procedures. Raw LC-MS metabolomics data were analysed using the open source software, IDEOM (http://mzmatch.sourceforge.net/ideom.php). Briefly, the IDEOM workflow uses msconvert to convert raw files to mzXML format, XCMS (Centwave) to pick LC-MS peak signals, and MZmatch for alignment and annotation of related metabolite peaks. Default IDEOM parameters were used to eliminate unwanted noise and artifact peaks. Confident metabolite identification was made by matching accurate masses to the retention time of the ~350 authentic standards. When these authentic standards were unavailable, putative metabolite identification used accurate mass and predicted retention times, as previously described (Creek et al. 2016). Metabolite abundance was represented by LC-MS peak height |
| Ion Mode: | POSITIVE |
| MS ID: | MS005578 |
| Analysis ID: | AN005858 |
| Instrument Name: | Thermo Orbitrap Exploris 120 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Data were acquired as a full scan in positive and negative ionization modes with a heated electrospray source and an Orbitrap resolution of 120,000 from 70 to 1,050 m/z. Ion source voltage was 3,500 V in positive mode and 2,500 V in negative mode. The ion transfer tube temperature was 325 °C and the vaporizer temperature was 350 °C. Gas mode was set to static with sheath gas, aux gas, and sweep gas at 50, 10, and 1, respectively. Samples within the LC-MS batch were sorted according to blocks of replicates and randomized. To facilitate metabolite identification, approximately 350 authentic metabolite standards were analyzed before the LC-MS batch, and their peaks and retention time were manually checked using the MZmine software. Pooled biological quality control samples and extraction solvent blanks were analyzed periodically throughout the batch to monitor LC-MS signal reproducibility and assist metabolite identification procedures. Raw LC-MS metabolomics data were analysed using the open source software, IDEOM (http://mzmatch.sourceforge.net/ideom.php). Briefly, the IDEOM workflow uses msconvert to convert raw files to mzXML format, XCMS (Centwave) to pick LC-MS peak signals, and MZmatch for alignment and annotation of related metabolite peaks. Default IDEOM parameters were used to eliminate unwanted noise and artifact peaks. Confident metabolite identification was made by matching accurate masses to the retention time of the ~350 authentic standards. When these authentic standards were unavailable, putative metabolite identification used accurate mass and predicted retention times, as previously described (Creek et al. 2016). Metabolite abundance was represented by LC-MS peak height |
| Ion Mode: | NEGATIVE |
