Identification of novel vulnerable antimalarial targets
Ontology highlight
ABSTRACT:
ORGANISM(S): Plasmodium falciparum
PROVIDER: GSE280287 | GEO | 2026/08/08
REPOSITORIES: GEO
Similar Datasets
Project description:Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3M-bM-^@M-2 untranslated region (3M-bM-^@M-2-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery. mRNA profiles were generated from 3D7 wild-type and DHFR-TS-GFP_glmS integrant parasites in untreated and treated with 10 mM Glucosamine conditions in duplicate.
2013-09-03 | E-GEOD-43125 | biostudies-arrayexpress
Project description:Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region (3′-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery.
2013-09-03 | GSE43125 | GEO
Project description:Identification of novel vulnerable antimalarial targets
| PRJNA1177753 | ENA
Project description:Like other functional RNAs, ribozymes contain a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-peripheral interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (ClvSeq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. Most deleterious mutations impaired RNA self-assembly. All-atom MD simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop rewire the network of hydrogen bonds around the catalytic site. Remarkably, IL4 mutations introduce a non-native helix interface that corrupts folding of the wild type core, eliminating activity. The results illustrate how competition between native and non-native structures in RNA drives the natural selection of central and peripheral tertiary interaction motifs.
2024-09-30 | GSE261357 | GEO
Project description:Plasmodium falciparum causes the most lethal form of malaria. The frontline treatments for this severe disease are combination therapies based on semisynthetic peroxide antimalarials, known as artemisinins. There is growing resistance to artemisinins and new drugs with novel mechanisms of action are urgently required. Synthetic peroxide antimalarials, known as ozonides, exhibit potent antimalarial activity both in vitro and in vivo. Here, we used chemical proteomics to investigate the protein alkylation targets of clickable artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, OZ439. We greatly expanded the list of protein targets for peroxide antimalarials and identified redox processes as being significantly enriched from the list of protein targets for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed with the use of a genetically encoded fluorescence-based biosensor comprising a redox-sensitive GFP (roGFP) fused to human glutaredoxin 1. This facilitated specific and dynamic live imaging of the glutathione redox potential in the cytosol of peroxide-treated infected red blood cells with high sensitivity and temporal resolution. We also used a targeted LC-MS based thiol metabolomics assay to accurately measure relative changes in cellular thiol levels (including thiol metabolites, glutathione precursors and oxidised and reduced glutathione) within peroxide-treated P. falciparum-infected red blood cells. 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.
2022-02-17 | PXD027334 | Pride
Project description:Drug resistance to nearly all antimalarials following their rollout underscores the need for novel chemotypes with novel mode of action to replenish the antimalarial drug-development pipeline. We identified a novel class of compounds in the antimalarial armory. Compound 31, characterized by a hydroxybenzamide scaffold, displays potent activity against blood-stage and late sexual stages of Plasmodium falciparum and no toxicity in human cells. Resistance selection studies with 31 identified a novel point mutation in the P. falciparum multidrug-resistance protein 1 (pfmdr1) gene, which was confirmed by CRISPR/Cas9-based gene editing as the primary mediator of resistance. Despite this, no cross-resistance towards first-line antimalarials were identified. Proteomics studies indicated that the primary mode of action of 31 is through direct binding to cytosolic ribosomal subunits, thereby inhibiting protein synthesis in the parasite. Taken together, compound 31 is a promising starting point for the development of a next-generation antimalarial.
2025-12-08 | PXD065399 | Pride
Project description:Inducible knockdown of Plasmodium gene expression using the glmS ribozyme
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Project description:The MORC (microrchidia) family of proteins is highly conserved in all eukaryotic cells and have been shown to play diverse roles by forming protein-protein interactions with immune-responsive proteins, SWI chromatin remodeling complexes, histone deacetylases, and histone tail modifications across metazoans. To dissect the functional roles of MORC in the human malaria parasite, Plasmodium falciparum, we developed a glmS based ribozyme knockdown system to induce PfMORC knockdown upon glucosamine (GlcN) induction. We further conducted a transcriptomic analysis in PfMORC-HA-glmS knockdown parasites at the asexual stage to investigate alterations in global gene expression. Our results indicate an overrepresentation of downregulated genes belonging to the heterochromatin-associated hypervariable gene family proteins. Overall, we found that PfMORC controls the asexual gene expression of the P. falciparum and can be a potential target for malaria disease containment.
2023-11-15 | GSE241313 | GEO
Project description: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.
2025-11-25 | PXD071156 | Pride
Project description:Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme
| PRJNA1086553 | ENA