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Validation of Solvent Proteome Profiling for Antimalarial Drug Target Deconvolution

ABSTRACT: Malaria remains a global health threat, with rising drug resistance accelerating the urgent need for new therapeutics. Target elucidation is a critical step in antimalarial drug discovery, enabling a deeper understanding of the molecular mechanisms of action of both existing and novel compounds. This study validates solvent-induced proteome profiling (SPP) as a proteomics-based approach for identifying drug-protein interactions in Plasmodium falciparum. SPP de-tects shifts in protein stability induced by ligand binding, allowing the identification of drug target/s without the need for compound modifications. Here, we successfully generated solvent denaturation curves for the P. falciparum pro-teome, and demonstrated the utility of SPP with five antimalarial compounds: pyrimethamine, atovaquone, cipar-gamin, MMV1557817 and OSM-S-106. In addition to measuring each compound’s impact across the full denatura-tion curve, investigating protein levels at individual solvent percentages preserved specific stability changes that would otherwise be masked in pooled analyses performed by integral SPP. This strategy was critical for the identifi-cation of the cipargamin target, non-SERCA-type Ca2+-transporting P-ATPase (PfATP4). Notably, we propose live-cell treatment SPP as a novel approach, demonstrating its ability to identify the validated target of pyrimethamine, bifunctional dihydrofolate reductase-thymidylate synthase (PfDHFR), with high specificity. We also introduced the novel one-pot mixed-drug SPP, which enables the evaluation of multiple drugs within a single lysate and experi-mental setup. This alternative method simplifies the experimental workflow and includes positive controls to affirm the performance of the experiment. Overall, this study demonstrates that SPP can be successfully applied in both ly-sate and live-cell treatment conditions to elucidate drug targets in P. falciparum, as well as providing additional in-formation regarding the mechanisms of drug action, offering insights for the optimisation of existing antimalarials and the development of novel therapies.

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ORGANISM(S): Plasmodium Falciparum (isolate 3d7)

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Yijia Ji

LAB HEAD: Darren J Creek

PROVIDER: PXD071156 | Pride | 2025-11-25

REPOSITORIES: Pride

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Project description:To determine the transcriptional effects of a novel plant-based compound, dehydrobrachylaenolide, on P. falciparum, parasite cultures were treated with the compound over time. Samples were taken for analysis 2, 6, and 12 hours post-invasion of human red blood cells. Control cultures were treated simultaneously with DMSO, and samples isolated at 2, 6, and 12 hours for transcriptional analysis. Background Antimalarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new antiplasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile. Methods Standard phytochemical analysis techniques including solvent-solvent extraction, thin-layer and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallised pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase assay. The effects of treatment on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls) with the pure compound, followed by oligonucleotide microarray and data analysis. Results The main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 245.6 nM, which was comparable to the IC50 of chloroquine, against a chloroquine-resistant strain (K1) of P. falciparum. Microarray data analysis identified a cluster of unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. This suggests differentiated modes of action between the two compounds. Conclusions Dehydrobrachylaenolide could play a valuable role as a drug candidate to generate new antimalarial compounds with novel modes of action and favourable ADMET properties.

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	ASTGS20240606_DM13_1.raw	Raw
	ASTGS20240606_DM13_2.raw	Raw
	ASTGS20240606_DM15_1.raw	Raw
	ASTGS20240606_DM15_2.raw	Raw
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Validation of Solvent Proteome Profiling for Antimalarial Drug Target Deconvolution

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