Biology subjects

Geoghegan, N. D.

Publications and source records attributed to Geoghegan, N. D..

6 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

SMCHD1's DNA binding activity enables its stable retention on chromatin

Chromatin proteins play critical roles in gene regulation, yet frequently we do not fully understand how weak DNA binding affinity of such proteins contributes to their locus-specific actions. Here, we studied SMCHD1, a non-canonical SMC-family protein involved in three-dimensional genome organization and gene repression of the inactive X chromosome and its autosomal targets. We replaced endogenous SMCHD1 with GFP-tagged wild-type or hinge-domain DNA-binding mutant SMCHD1 to define the cellular role of DNA binding. The mutant showed reduced enrichment at the inactive X chromosome in female cells, while retaining stable binding at most autosomal binding sites. Impaired DNA binding weakens SMCHD1-mediated gene repression and chromatin-state regulation, producing hypomorphic effect. Multiple live-cell imaging methods reveal that DNA binding constrains SMCHD1 mobility and supports maintenance, rather than initial recruitment, of chromatin-bound SMCHD1 both during interphase and mitosis. Thus, SMCHD1's weak and sequence-independent DNA binding is a key determinant of its chromatin residence, localization and function. Our findings provide a framework for understanding SMCHD1 and other chromatin proteins with sequence-independent DNA binding activity.

genomics↗

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

Global analysis of cancer cell responses to USP9X inhibition

The ubiquitin specific protease (USP) enzyme USP9X is amongst the best studied human deubiquitinases (DUBs), with a myriad of described targets and cellular roles. In cancer, USP9X has been touted as both an oncogene and a tumour suppressor in different contexts, which has confounded the field and questioned its viability as a cancer target. We here describe WEHI-092, a novel piperazine-based USP9X specific small molecule inhibitor and map its binding site to a unique region in the USP9X fingers subdomain, distinct from known DUB inhibitor binding sites. Using proteomics and ubiquitinomics, we show that USP9X has a distinct set of substrates compared to USP7 indicating remarkable DUB target specificity, yet the substrate profile of USP9X varies significantly across cancer cell lines. Interestingly, we reveal a core set of 17 proteins commonly regulated by USP9X in most or all cell lines, which we consider as proximal biomarkers for USP9X inhibition. Consistent with our proteomic analyses, we show that WEHI-092 treatment arrests cells in metaphase without inducing cell death, which may account for growth suppression seen in long-term clonogenic assays in most cancer cell lines, and positions USP9X inhibitors as a new potential class of selective mitotic poisons.

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↗