Summary of Study ST001175

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 PR000787. The data can be accessed directly via it's Project DOI: 10.21228/M8Z97B This work is supported by NIH grant, U2C- DK119886.

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

Study ID	ST001175
Study Title	Multi-omics analysis demonstrates unique mode of action of a potent new antimalarial compound, JPC-3210, against Plasmodium falciparum
Study Summary	The increasing incidence of antimalarial drug resistance to the first-line artemisinins, and their combination partner drugs, underpins an urgent need for new antimalarial drugs, ideally with a novel mechanism of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum, low cytotoxicity, potent in vivo efficacy against murine malaria, and favourable preclinical pharmacokinetics, including a lengthy plasma elimination half-life. This study demonstrates the application of a “multi-omics” workflow based on high resolution orbitrap mass spectrometry to investigate the impact of JPC-3210 on biochemical pathways within P. falciparum infected red blood cells. Metabolomics and peptidomics analysis revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a depletion in short hemoglobin-derived peptides, while peptidomics analysis showed a depletion in longer hemoglobin-derived peptides. In order to further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerisation assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerisation, about 10-fold less potent then the quinoline antimalarials. Furthermore, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature in comparison to other known antimalarials. Whilst JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. In conclusion, multi-omics studies using high resolution mass spectrometry revealed JPC-3210 to possess a unique mechanism of action involving inhibition of hemoglobin digestion, depletion of DNA replication and synthesis proteins, and elevation of regulators of protein translation. Importantly, this mechanism is distinct from currently-used antimalarials, suggesting that JPC-3210 warrants further investigation as a potentially useful new antimalarial agent.
Institute	Monash University
Last Name	Siddiqui
First Name	Ghizal
Address	381 Royal Parade, Parkville
Email	ghizal.siddiqui@monash.edu
Phone	99039282
Submit Date	2019-04-25
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2019-05-15
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP001249
Sampleprep Summary:	Following drug incubation, metabolites were extracted as detailed. In short, after incubation with drug, culture medium was aspirated from each well, and the metabolism of infected red blood cells (iRBCs) was quenched by the addition of ice-cold phosphate-buffered saline (PBS). Cells were pelleted by centrifugation for 5 min at 1,000 × g, and the PBS supernatant was removed prior to the addition of 135 μL ice cold methanol (containing the internal standard compounds; CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS [N-cyclohexyl-3-aminopropane-sulfonic acid], and PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]). Samples were rapidly mixed by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 min and then centrifuged at 3,000 × g to remove insoluble material. Supernatants were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80°C until analysis. An aliquot (10 μL) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor downstream sample stability and analytical reproducibility.

Sampleprep ID:

SP001249

Sampleprep Summary:

Following drug incubation, metabolites were extracted as detailed. In short, after incubation with drug, culture medium was aspirated from each well, and the metabolism of infected red blood cells (iRBCs) was quenched by the addition of ice-cold phosphate-buffered saline (PBS). Cells were pelleted by centrifugation for 5 min at 1,000 × g, and the PBS supernatant was removed prior to the addition of 135 μL ice cold methanol (containing the internal standard compounds; CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS [N-cyclohexyl-3-aminopropane-sulfonic acid], and PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)]). Samples were rapidly mixed by pipetting three times to extract iRBC metabolites. Samples were left on ice with gentle agitation for 60 min and then centrifuged at 3,000 × g to remove insoluble material. Supernatants were transferred to glass high-performance liquid chromatography (HPLC) vials and stored at −80°C until analysis. An aliquot (10 μL) of each sample was combined to generate a pooled biological quality control (PBQC) sample, which was used to monitor downstream sample stability and analytical reproducibility.