Biology subjects

Sleebs, B. E.

Publications and source records attributed to Sleebs, B. E..

9 recordsLinked to original sources

bioRxiv2026

Plasmodium berghei is resistant to aryl amino acetamides that inhibit P. falciparum growth by targeting the phospholipid transfer protein PfSTART1.

In a previous screen for compounds that inhibit Plasmodium falciparum merozoite invasion of red blood cells, we identified the Medicines for Malaria Venture compound MMV006833. This compound inhibits PfSTART1, a protein implicated in the expansion of the nascent parasitophorous vacuole membrane following invasion, to accommodate the developing ring-stage parasite. Live-cell lattice light-sheet microscopy of invading merozoites revealed that mNeonGreen-tagged PfSTART1 is released from structures within the merozoite into the nascent parasitophorous vacuole approximately 109 seconds after invasion. Expansion microscopy of PfSTART1-HA merozoites further showed that these punctate PfSTART1-containing structures do not colocalise with known secretory organelles (rhoptries, micronemes and dense granules). Although analogues of MMV006833 are highly potent against P. falciparum, they were previously found to be ineffective against P. berghei parasites in the mouse malaria model. Here, we demonstrate that PbSTART1 is highly resistant to MMV006833 and its analogues when expressed in P. falciparum, indicating that structural differences between the orthologous proteins reduce inhibitor potency. The crystal structure of PfSTART1 in complex with WEHI-991 revealed the molecular basis for inhibition and provided a structural explanation for the reduced potency of this family of compounds against P. berghei. To sensitise P. berghei parasites to MMV006833 analogues, the parasites were engineered to express PfSTART1; however, these chimeric parasites remained insensitive to the compounds. This suggests that factors beyond target engagement, such as compound half-life or bioavailability, contribute to the lack of efficacy observed in the mouse malaria model.

microbiology↗

bioRxiv2026

Formation of the moving junction is the nexus for host cytoskeletal remodelling during Plasmodium falciparum invasion of human erythrocytes

Plasmodium falciparum invasion of human erythrocytes is a complex and tightly coordinated process, involving host cell attachment, moving junction formation and engagement of the parasites actomyosin motor. The temporal precision of these events is mediated by distinct ligand-receptor interactions and the sequential release of the merozoites apical organelles. What remains unclear is how these molecular and biophysical interactions enable Plasmodium to bypass the stable erythrocyte membrane-cytoskeletal complex. Here, several P. falciparum lines expressing different fluorescently tagged apical organelle proteins, were imaged with lattice light sheet microscopy (LLSM) to determine the timing of cytoskeletal disassembly and apical organelle release. Blocking the AMA1-RON2 interaction has no effect on the PfRh5-basigin Ca2+ flux but prevents host cytoskeleton disassembly. In contrast, the inhibition of parasite actin polymerisation had no effect on cytoskeletal clearance but caused a sustained Ca2+ response. We further demonstrate that establishment of the moving junction is temporally linked to clearance of the host cytoskeleton. Collectively, our findings support the existence of an association between the RON complex and components of the host cytoskeleton, which mediates the localised disruption of the erythrocyte-membrane cytoskeletal complex during invasion.

microbiology↗

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

Deconvolution of the on-target activity of plasmepsin V peptidomimetics in P. falciparum parasites

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of Plasmodium by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a bona fide antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent induced protein precipitation (SIP) and intact-cell thermal PISA profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant (SNV) within the pmv gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

cell biology↗

bioRxiv2023

Aryl amino acetamides prevent the development of Plasmodium falciparum rings via inhibition of the lipid transfer protein PfSTART1

With resistance to most antimalarials increasing, it is imperative that new antimalarial drugs are developed to replace or complement front-line artemisinin therapies. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited ring development of newly invaded merozoites. Here, we selected parasites resistant to M-833 and identified independent mutations arising in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introduction of the identified PfSTART1 mutations into wildtype parasites reproduced resistance to both M-833 and highly potent analogues, confirming PfSTART1 mutations were sufficient to confer resistance. The analogues bound to recombinant PfSTART1 with nanomolar affinity. We also demonstrated selective PfSTART1 engagement by the analogues using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assay for the first time in Plasmodium. Imaging of newly invaded merozoites showed the inhibitors prevented the conversion into larger amoeboid ring-stage parasites potentially through the inhibition of phospholipid transfer from the parasite to the encasing parasitophorous vacuole membrane (PVM) and/or within the parasite. We show that these PfSTART1 inhibitors also block transmission. With multiple stages of the parasites lifecycle being targeted by PfSTART1 inhibitors, this protein therefore represents a novel drug target with a new mechanism of action.

cell biology↗

bioRxiv2023

A pyridyl-furan series developed from Open Global Health Library blocks red blood cell invasion and protein trafficking in Plasmodium falciparum through potential inhibition of the parasites PI4KIIIb enzyme.

With resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250, was prioritised for further optimisation. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasites life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB which clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies which showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

microbiology↗

bioRxiv2022

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasite Plasmodium falciparum through interference with actin-1/profilin dynamics

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro co-sedimentation experiments using recombinant P. falciparum actin-1 and profilin proteins indicate that potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, thereby reducing the formation of filamentous actin. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

molecular biology↗

bioRxiv2022

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current anti-malarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria box of 400 anti-malarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs - modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future anti-malarial that rapidly inhibits parasite virulence before eliminating parasites from the host. SynopsisPlasmodium falciparum exports proteins into its host cell to perform a myriad of functions required for survival. We adapted an assay to screen for small molecules that inhibit protein secretion and export. Screening the 400-compound Medicines for Malaria Venture (MMV) Malaria Box uncovered several potential export inhibitors. The most promising of these compounds, MMV396797, blocked protein export at the parasite and reduced host rigidification and cytoadherence, two functions which are mediated by exported proteins.

biochemistry↗

bioRxiv2019

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

To identify potential inhibitors of egress and invasion in the asexual blood stage of Plasmodium falciparum, we screened the Medicines for Malaria Venture (MMV) Pathogen Box. This compound library comprises of 400 drugs against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, Nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 {micro}M, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has greater implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.\n\nImportancePlasmodium falciparum causes the most severe form of malaria and with emerging resistance to frontline treatments, there is the need to identify new drug targets in the parasite. One of the most critical processes during the asexual blood stage in the parasites lifecycle is the egress from old red blood cells (RBCs) and subsequent invasion of new RBCs. Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. Identifying novel compounds that inhibit these essential processes would further their development into possible antimalarials that would be highly effective at killing asexual RBC stage parasites when used in combination with drugs that target the intraerythrocytic growth phase. These compounds potentially may also be used as novel tools to study the complex biology of parasites to gain further insight into the mechanisms behind egress and invasion.

cell biology↗