Summary of Study ST003179

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 PR001978. The data can be accessed directly via it's Project DOI: 10.21228/M8214C 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	ST003179
Study Title	Property and Activity Refinement of Dihydroquinazolinone-3-carboxamides as Orally Efficacious Antimalarials that Target PfATP4
Study Summary	The development of new antimalarial classes is pertinent because of resistance against the current antimalarial treatments. To contribute to the global effort to create new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. The preliminary optimization generated analogs, such as 4, that exhibited potent in vitro asexual stage activity but only showed modest oral efficacy in a P. berghei mouse model attributed to its low aqueous solubility and modest metabolic stability. Here we report on correcting these parameters to improve in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold markedly improved aqueous solubility without a significant loss of asexual parasite activity. Certain configurations of pyrazoles in the 8-position were found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. We were able to show that modifications made to the optimized analogs, such as WJM992 did not perturb the sensitivity to PfATP4 drug-resistant parasites or alter on-target activity in a PfATP4-associated parasite cytosolic Na+ flux assay and gave a distinct metabolic signature indicative of other PfATP4 inhibitors. The optimized analogs showed an appreciable efficacy in malaria mouse models and blocked sexual stage gamete development preventing transmission to mosquitoes.
Institute	Monash University
Last Name	Giannangelo
First Name	Carlo
Address	381 Royal Parade, Parkville, Victoria, 3052, Australia
Email	carlo.giannangelo@monash.edu
Phone	99039282
Submit Date	2024-04-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-05-15
Release Version	1

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Project:

Project ID:	PR001978
Project DOI:	doi: 10.21228/M8214C
Project Title:	Property and Activity Refinement of Dihydroquinazolinone-3-carboxamides as Orally Efficacious Antimalarials that Target PfATP4
Project Summary:	The development of new antimalarial classes is pertinent because of resistance against the current antimalarial treatments. To contribute to the global effort to create new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. The preliminary optimization generated analogs, such as 4, that exhibited potent in vitro asexual stage activity but only showed modest oral efficacy in a P. berghei mouse model attributed to its low aqueous solubility and modest metabolic stability. Here we report on correcting these parameters to improve in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold markedly improved aqueous solubility without a significant loss of asexual parasite activity. Certain configurations of pyrazoles in the 8-position were found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. We were able to show that modifications made to the optimized analogs, such as WJM992 did not perturb the sensitivity to PfATP4 drug-resistant parasites or alter on-target activity in a PfATP4-associated parasite cytosolic Na+ flux assay and gave a distinct metabolic signature indicative of other PfATP4 inhibitors. The optimized analogs showed an appreciable efficacy in malaria mouse models and blocked sexual stage gamete development preventing transmission to mosquitoes.
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:	SU003298
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833

Factors:

Subject type: Cultured cells; Subject species: Plasmodium falciparum (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA345846	DMSO_1	Plasmodium cells	DMSO
SA345847	DMSO_4	Plasmodium cells	DMSO
SA345848	DMSO_2	Plasmodium cells	DMSO
SA345849	DMSO_3	Plasmodium cells	DMSO
SA345850	KAE609_20x_1	Plasmodium cells	KAE609_20x_IC50
SA345851	KAE609_20x_3	Plasmodium cells	KAE609_20x_IC50
SA345852	KAE609_20x_2	Plasmodium cells	KAE609_20x_IC50
SA345853	KAE609_5x_3	Plasmodium cells	KAE609_5x_IC50
SA345854	KAE609_5x_2	Plasmodium cells	KAE609_5x_IC50
SA345855	KAE609_5x_1	Plasmodium cells	KAE609_5x_IC50
SA345856	WJM992_3	Plasmodium cells	WJM992
SA345857	WJM992_2	Plasmodium cells	WJM992
SA345858	WJM992_1	Plasmodium cells	WJM992

Showing results 1 to 13 of 13

Showing results 1 to 13 of 13

Collection:

Collection ID:	CO003291
Collection Summary:	Samples were collected from P. falciparum cultures (3D7 strain) synchronized to the mid trophozoite stage (28-36 hours post invasion). They were magnet purified to achieve a parasitemia of >90% and hematocrit of 0.5%. The culture medium was refreshed immediately before compound incubation.
Sample Type:	Plasmodium cells

Treatment:

Treatment ID:	TR003307
Treatment Summary:	Infected red blood cells were treated with 70 nM of compound 10ah (5 x EC50), 5 nM (5 x EC50), or 20 nM (20 x EC50) of the known PfATP4 inhibitor KAE609 1, or an equivalent volume of vehicle (DMSO) for 5 h with a minimum of three independent incubations per condition.

Sample Preparation:

Sampleprep ID:	SP003305
Sampleprep Summary:	Following compound incubation, cultures were centrifuged at 1,200 g for 3 min, the media was removed, and the cell pellets were washed in 1 mL of ice-cold PBS. Samples were again centrifuged at 1,200 g for 3 min to remove all of the PBS and metabolites were extracted from 5 x 107 cells using 90 µL of ice-cold methanol extraction solvent. Samples were then incubated on an automatic vortex mixer at 4 °C for 1 h before being centrifuged at 21,000 g for 10 min. The supernatants were transferred into high-performance liquid chromatography (HPLC) vials and stored 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.

Sampleprep ID:

SP003305

Sampleprep Summary:

Following compound incubation, cultures were centrifuged at 1,200 g for 3 min, the media was removed, and the cell pellets were washed in 1 mL of ice-cold PBS. Samples were again centrifuged at 1,200 g for 3 min to remove all of the PBS and metabolites were extracted from 5 x 107 cells using 90 µL of ice-cold methanol extraction solvent. Samples were then incubated on an automatic vortex mixer at 4 °C for 1 h before being centrifuged at 21,000 g for 10 min. The supernatants were transferred into high-performance liquid chromatography (HPLC) vials and stored 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	AN005221	AN005222
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Merck SeQuant ZIC-pHILIC (150 x 4.6mm,5um)	Merck SeQuant ZIC-pHILIC (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:	CH003949
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-pHILIC (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:	MS004954
Analysis ID:	AN005221
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. Metabolite abundance was represented by LC-MS peak height.
Ion Mode:	POSITIVE

MS ID:	MS004955
Analysis ID:	AN005222
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. Metabolite abundance was represented by LC-MS peak height.
Ion Mode:	NEGATIVE