Summary of Study ST001315

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 PR000892. The data can be accessed directly via it's Project DOI: 10.21228/M8CX0M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001315
Study Title	Retargeting azithromycin-like compounds as antimalarials with dual modality
Study Summary	Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium parasites that cause malaria are urgently needed. Here, we show that azithromycin—a clinically used macrolide antibiotic that targets the bacterium-like ribosome of the malaria parasites apicoplast organelle and causes a slow-killing ‘delayed death’ phenotype—can also rapidly kill parasites throughout the asexual blood-stages of the lifecycle via a ‘quick-killing’ mechanism of action. Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency that is directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin for in vitro treatment windows less than 48 hours. Analogues were also effective against the zoonotic malaria parasite P. knowlesi, and against both multi-drug and artemisinin resistant P. falciparum lines. Metabolomic profiles of azithromycin analogue treated parasites were similar to those of chloroquine treated parasites, suggesting that the quick-killing mechanism of action may in part be localised to the parasite food vacuole. However, metabolomic signatures associated with mitochondrial disruption were also present. In addition, unlike chloroquine, azithromycin and analogues were active across blood stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on quick-killing activity, which suggests that apicoplast-targeting, delayed-death activity can either be preserved or removed independently of quick-killing. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Therefore, development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed death mechanism of action in a single, multifactorial chemotype.
Institute	Monash University
Last Name	Siddiqui
First Name	Ghizal
Address	381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Email	ghizal.siddiqui@monash.edu
Phone	99039282
Submit Date	2020-02-06
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2020-03-03
Release Version	1

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Project:

Project ID:	PR000892
Project DOI:	doi: 10.21228/M8CX0M
Project Title:	Retargeting azithromycin-like compounds as antimalarials with dual modality
Project Summary:	Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium parasites that cause malaria are urgently needed. Here, we show that azithromycin—a clinically used macrolide antibiotic that targets the bacterium-like ribosome of the malaria parasites apicoplast organelle and causes a slow-killing ‘delayed death’ phenotype—can also rapidly kill parasites throughout the asexual blood-stages of the lifecycle via a ‘quick-killing’ mechanism of action. Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency that is directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin for in vitro treatment windows less than 48 hours. Analogues were also effective against the zoonotic malaria parasite P. knowlesi, and against both multi-drug and artemisinin resistant P. falciparum lines. Metabolomic profiles of azithromycin analogue treated parasites were similar to those of chloroquine treated parasites, suggesting that the quick-killing mechanism of action may in part be localised to the parasite food vacuole. However, metabolomic signatures associated with mitochondrial disruption were also present. In addition, unlike chloroquine, azithromycin and analogues were active across blood stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on quick-killing activity, which suggests that apicoplast-targeting, delayed-death activity can either be preserved or removed independently of quick-killing. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Therefore, development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed death mechanism of action in a single, multifactorial chemotype.
Institute:	Monash University
Last Name:	Siddiqui
First Name:	Ghizal
Address:	381 Royal Parade, Parkville, Melbourne, Victoria, 3052, Australia
Email:	ghizal.siddiqui@monash.edu
Phone:	99039282

Subject:

Subject ID:	SU001389
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	treatment	Experiment
SA094772	Az_E1_1	azithromycin	1
SA094773	Az_E1_2	azithromycin	1
SA094774	Az_E1_3	azithromycin	1
SA094775	Az_E2_3	azithromycin	2
SA094776	Az_E2_1	azithromycin	2
SA094777	Az_E2_2	azithromycin	2
SA094736	CQ_E1_1	Chloroquine	1
SA094737	CQ_E1_3	Chloroquine	1
SA094738	CQ_E1_2	Chloroquine	1
SA094739	CQ_E2_2	Chloroquine	2
SA094740	CQ_E2_1	Chloroquine	2
SA094741	CQ_E2_3	Chloroquine	2
SA094742	DHA_E1_2	Dihydroartemisinin	1
SA094743	DHA_E1_1	Dihydroartemisinin	1
SA094744	DHA_E1_3	Dihydroartemisinin	1
SA094745	DHA_E2_3	Dihydroartemisinin	2
SA094746	DHA_E2_2	Dihydroartemisinin	2
SA094747	DHA_E2_1	Dihydroartemisinin	2
SA094748	Control_E1_1	Ethanol control	1
SA094749	Control_E1_3	Ethanol control	1
SA094750	Control_E1_2	Ethanol control	1
SA094751	Control_E2_1	Ethanol control	2
SA094752	Control_E2_2	Ethanol control	2
SA094753	Control_E2_3	Ethanol control	2
SA094754	GSK5_E1_1	GSK5	1
SA094755	GSK5_E1_2	GSK5	1
SA094756	GSK5_E1_3	GSK5	1
SA094757	GSK5_E2_3	GSK5	2
SA094758	GSK5_E2_1	GSK5	2
SA094759	GSK5_E2_2	GSK5	2
SA094760	GSK66_E1_1	GSK66	1
SA094761	GSK66_E1_2	GSK66	1
SA094762	GSK66_E1_3	GSK66	1
SA094763	GSK66_E2_2	GSK66	2
SA094764	GSK66_E2_3	GSK66	2
SA094765	GSK66_E2_1	GSK66	2
SA094766	GSK71_E1_3	GSK71	1
SA094767	GSK71_E1_1	GSK71	1
SA094768	GSK71_E1_2	GSK71	1
SA094769	GSK71_E2_1	GSK71	2
SA094770	GSK71_E2_3	GSK71	2
SA094771	GSK71_E2_2	GSK71	2

Showing results 1 to 42 of 42

Showing results 1 to 42 of 42

Collection:

Collection ID:	CO001384
Collection Summary:	For metabolomics experiments, two 150 mL flasks at 6% haematocrit containing tightly synchronised ~30-34 hr trophozoites were harvested via magnet purification (Miltenyi Biotech). Infected RBC density was quantitated by flow cytometry and 2 mL of 3x 107 parasites were added to and incubated in 24 well microtiter plates for 1 hr at 37oC to stabilise the culture. Drugs (5x IC50) were added and incubated for a further 2 hrs prior to removal of the supernatant, 2x washes with 800 L ice-cold 1 x PBS with cells pelleted via centrifugation at 400 x g for 5 mins at 4oC. The cell pellets were resuspended in 150 L of ice-cold extraction buffer (MeOH) containing 1 µM internal standards; CHAPS and PIPES, and incubated on ice for 1 hr with shaking at 200 rpm. Insoluble material was pelleted with centrifugation at 14,800 x g for 10 mins at 4 oC and 120 µL of supernatant was collected and stored at -80 ºC until analysis.
Sample Type:	Blood (whole)

Treatment:

Treatment ID:	TR001404
Treatment Summary:	Drugs (5x IC50) (azithromycin, dihydroartemisinin, chloroquine, GSK-5, GSK71, GSK-66 and ethanol control) were added and incubated for a further 2 hrs prior to removal of the supernatant

Sample Preparation:

Sampleprep ID:	SP001397
Sampleprep Summary:	2x washes with 800 L ice-cold 1 x PBS with cells pelleted via centrifugation at 400 x g for 5 mins at 4oC. The cell pellets were resuspended in 150 L of ice-cold extraction buffer (MeOH) containing 1 µM internal standards; CHAPS and PIPES, and incubated on ice for 1 hr with shaking at 200 rpm. Insoluble material was pelleted with centrifugation at 14,800 x g for 10 mins at 4 oC and 120 µL of supernatant was collected and stored at -80 ºC until analysis.

Sampleprep ID:

SP001397

Sampleprep Summary:

2x washes with 800 L ice-cold 1 x PBS with cells pelleted via centrifugation at 400 x g for 5 mins at 4oC. The cell pellets were resuspended in 150 L of ice-cold extraction buffer (MeOH) containing 1 µM internal standards; CHAPS and PIPES, and incubated on ice for 1 hr with shaking at 200 rpm. Insoluble material was pelleted with centrifugation at 14,800 x g for 10 mins at 4 oC and 120 µL of supernatant was collected and stored at -80 ºC until analysis.

Combined analysis:

Analysis ID	AN002189	AN002190
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Signal Intensity	Signal Intensity

Chromatography:

Chromatography ID:	CH001604
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
Column Temperature:	25
Flow Gradient:	80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min.
Flow Rate:	0.3 ml/min
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

Chromatography ID:	CH001605
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	SeQuant ZIC-pHILIC (150 x 4.6mm,5um)
Column Temperature:	25
Flow Gradient:	80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min.
Flow Rate:	0.3 ml/min
Solvent A:	100% water; 20 mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS002036
Analysis ID:	AN002189
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Liquid chromatography-mass spectrometry (LC-MS) data was acquired on a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled with high-performance liquid chromatography system (HPLC, Dionex Ultimate® 3000 RS, Thermo Scientific) as per previously described 49. Briefly, chromatographic separation was performed on a ZIC-pHILIC column equipped with a guard (5 µm, 4.6 × 150 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate (Sigma Aldrich), (B) acetonitrile (Burdick and Jackson) and needle wash solution was 50% isopropanol. The column flow rate was maintained at 0.3 ml/min with temperature at 25 ºC and the gradient program was as follows: 80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min. Total run time was 32 min with an injection volume of 10 µL. Mass spectrometer was operated in full scan mode with positive and negative polarity switching at 35k resolution at 200 m/z, with detection range of 85 to 1275 m/z, AGC target was 1e6 ions with a maximum injection time of 50 ms. Electro-spray ionization source (HESI) was set to 4.0 kV voltage for positive and negative mode, sheath gas was set to 50, aux gas to 20 and sweep gas to 2 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. The samples were analyzed as a single batch to avoid batch-to-batch variation and randomized to account for LCMS system drift over time. Repeated analysis of pooled quality control samples was performed throughout the batch to confirm signal reproducibility.
Ion Mode:	POSITIVE

MS ID:	MS002037
Analysis ID:	AN002190
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Liquid chromatography-mass spectrometry (LC-MS) data was acquired on a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled with high-performance liquid chromatography system (HPLC, Dionex Ultimate® 3000 RS, Thermo Scientific) as per previously described 49. Briefly, chromatographic separation was performed on a ZIC-pHILIC column equipped with a guard (5 µm, 4.6 × 150 mm, SeQuant®, Merck). The mobile phase (A) was 20 mM ammonium carbonate (Sigma Aldrich), (B) acetonitrile (Burdick and Jackson) and needle wash solution was 50% isopropanol. The column flow rate was maintained at 0.3 ml/min with temperature at 25 ºC and the gradient program was as follows: 80% B decreasing to 50% B over 15 min, then to 5% B at 18 min until 21 min, increasing to 80% B at 24 min until 32 min. Total run time was 32 min with an injection volume of 10 µL. Mass spectrometer was operated in full scan mode with positive and negative polarity switching at 35k resolution at 200 m/z, with detection range of 85 to 1275 m/z, AGC target was 1e6 ions with a maximum injection time of 50 ms. Electro-spray ionization source (HESI) was set to 4.0 kV voltage for positive and negative mode, sheath gas was set to 50, aux gas to 20 and sweep gas to 2 arbitrary units, capillary temperature 300 °C, probe heater temperature 120 °C. The samples were analyzed as a single batch to avoid batch-to-batch variation and randomized to account for LCMS system drift over time. Repeated analysis of pooled quality control samples was performed throughout the batch to confirm signal reproducibility.
Ion Mode:	NEGATIVE