Biology subjects

MacRaild, C. A.

Publications and source records attributed to MacRaild, C. A..

4 recordsLinked to original sources

bioRxiv2026

MMV687794 blocks Plasmodium falciparum invasion of red blood cells by targeting a Surface-associated Lipid-Interacting Rhoptry Protein, PfSLIRP

Invasion of red blood cells (RBCs) by the human malaria parasite, Plasmodium falciparum, drives disease. During invasion, the parasite pushes its way into the RBC while wrapping the RBC membrane around itself to establish the parasitophorous vacuole, a stable niche within the RBC where the parasite grows. To better understand invasion, we investigated the mechanism of action of an invasion-inhibitory compound, MMV687794 (MMV794). Lattice light-sheet microscopy revealed that MMV794 blocks parasite entry by preventing parasitophorous vacuole formation. In vitro drug resistance selection of parasites with MMV794 found mutations to the /{beta} hydrolase, PF3D7_0403800, and engineering one of these mutations (C36W) into parasites by CRISPR/Cas9 recapitulated the resistance phenotype. Expansion microscopy demonstrated that this protein is expressed in schizonts, localising to the surface of rhoptries, which are specialised apical secretory organelles that function during invasion. Lipidomics and proteomics analyses of C36W parasites uncovered widespread changes to lipid composition/homeostasis and altered abundance of proteins involved in invasion, indicating a role for PF3D7_0403800 in invasion-associated lipid metabolism. Finally, we used solvent-induced proteome profiling and reactivity assays to confirm drug-target engagement. Together, our findings identify a novel Surface-associated Lipid-Interacting Rhoptry Protein (PfSLIRP) that coordinates lipid metabolism at the rhoptries to enable RBC invasion.

microbiology↗

bioRxiv2025

Validation of Solvent Proteome Profiling for Antimalarial Drug Target Deconvolution

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 detects 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 proteome, and demonstrated the utility of SPP with five antimalarial compounds: pyrimethamine, atovaquone, cipargamin, MMV1557817 and OSM-S-106. In addition to measuring each compounds impact across the full denaturation 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 identification 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 experimental 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 lysates and live cells to elucidate drug targets in P. falciparum, as well as providing additional information regarding the mechanisms of drug action, offering insights for the optimisation of existing antimalarials and the development of novel therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/670428v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b83c75org.highwire.dtl.DTLVardef@1895f23org.highwire.dtl.DTLVardef@1c51ba0org.highwire.dtl.DTLVardef@198c822_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2021

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway

Plasmodium falciparum, a causative agent of malaria, continues to remain a global health threat since these parasites have developed increasing resistance to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

microbiology↗

bioRxiv2021

Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials. ImportanceThe frontline treatments for malaria are combination therapies based on the peroxide antimalarial, artemisinin. Concerningly, artemisinin resistance has emerged in malaria-endemic regions, and now poses a major threat to malaria treatment and eradication efforts. New medicines are urgently required to replace the artemisinins, and some of the most advanced candidates are the fully synthetic peroxide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel). The mechanism of action of peroxide antimalarials involves the reductive activation of the peroxide bond by intra-parasitic haem, but there is no consensus regarding the specific protein targets of the resulting radical species for artemisinins and/or the ozonides. This study provides a comprehensive and unbiased chemoproteomic profile of over 400 target proteins, and confirms the specific impact of peroxide antimalarials on redox metabolism. The key role of redox targets is particularly relevant considering that the mechanism of artemisinin resistance appears to involve modulation of peroxide activation and redox homeostasis.

pharmacology and toxicology↗