Biology subjects

Boulet, C.

Publications and source records attributed to Boulet, C..

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↗

bioRxiv2025

Redefining the role of the Plasmodium heme detoxification protein: From hemozoin formation to mitochondrial protein synthesis

Throughout their intraerythrocytic development, malaria parasites digest up to 80% of the host cells hemoglobin within a specialized degradative compartment known as the digestive vacuole. This process releases heme, which is detoxified by sequestration into bioinert hemozoin crystals. Although heme biomineralization is essential for blood-stage survival and a validated drug target, its underlying mechanisms remain unclear. Initially identified as a potent inducer of {beta}-hematin crystallization in vitro, the parasites Heme Detoxification Protein (HDP) has been proposed to execute a similar role in the formation of hemozoin crystals in cellulo. Here, we investigate the function of HDP in live Plasmodium falciparum parasites, integrating experimental genetic approaches with quantitative microscopy, cellular bioenergetics and whole-proteome profiling. Endogenous tagging revealed that HDP localizes to the mitochondrion rather than the digestive vacuole. Conditional inactivation of HDP resulted in a gradual loss of mitochondrial membrane potential, preceding developmental arrest. Bypassing the essential role of the respiratory chain in pyrimidine biosynthesis - either through exogenous electron acceptors or expression of a ubiquinone-independent dihydroorotate dehydrogenase - rescued HDP-deficient parasites, indicating a role in maintaining respiratory chain activity. Consistent with this, electron flow through complex IV was abolished in rescued HDP-null parasites, rendering them hypersensitive to proguanil, an antimalarial that synergizes with respiratory chain inhibitors. We found that loss of HDP leads to a marked reduction of complexes III and IV, whose integrity depends on mitochondrial protein biosynthesis. Integration of quantitative proteomic data with structure-guided homology modelling supports a role for HDP as part of the large mitoribosomal subunit at the inter-subunit contact site. By contrast, HDP loss did not affect the quantity of hemozoin or other heme species, crystal morphology, or sensitivity to the hemozoin-targeting drug chloroquine. Together, these findings challenge previous models linking HDP to hemozoin formation and instead reveal an essential role for HDP in mitochondrial protein biosynthesis.

microbiology↗

bioRxiv2025

Targeting ligand binding sites in Plasmodium falciparum NCR1 enables antimalarial drug discovery

PfNCR1 is a Plasmodium falciparum cholesterol transporter at the plasma membrane-parasitophorous vacuole interface, which has recently emerged as a promising antimalarial target. Despite an immense interest in development of novel antimalarial compounds targeting PfNCR1, the molecular mechanism of PfNCR1 inhibition remains elusive. Here, we report cryo-EM structures of PfNCR1 in its apo state and bound to three inhibitors: MMV009108, MMV019662 and MMV028038. MMV009108 binds to the ''neck'' site at the ectodomain-membrane domain inter-face. MMV028038 displaces the sterol at the ectodomain ''ecto'' site. Remarkably, MMV019662 binds both sites: it associates near the bound sterol molecule at the ecto site and targets the neck site, thereby altering the sterol-sensing domain conformation. Importantly, we identify a novel antimalarial compound, G856-4236, which targets the ecto site exclusively. These four distinct modes of PfNCR1 inhibition advance our understanding of its conformational plasticity and es-tablish a framework for rational drug discovery targeting PfNCR1 and related transporters.

biochemistry↗

bioRxiv2025

Aryl N-acetamide compounds exert antimalarial activity by acting as agonists of rhomboid protease PfROM8 and cation channel PfCSC1.

With resistance to current frontline antimalarials spreading globally, new drug candidates need to be discovered to populate the antimalarial drug development pipeline. We previously screened the Medicines for Malaria Venture Pathogen Box for compounds that prevent Plasmodium falciparum parasites from exiting and invading human erythrocytes, steps essential for the proliferation of parasites in the blood, which causes disease. Compound MMV020512 (M-512) was identified in this screen and live cell imaging here established that it does not specifically inhibit invasion but likely inhibits intraerythrocytic parasite growth. M-512 resistance selection in parasites led to the identification of mutations in the membrane protease PfROM8 and the cation ion channel PfCSC1. PfROM8 was validated as a target of M-512 when a L562R putative resistance mutation was engineered into wildtype parasites reproducing the resistance phenotype. Knockdown of wildtype PfROM8, the L562R mutant and CSC1 reduced parasite growth, indicating the proteins are functionally important. Counterintuitively, the PfROM8 and PfCSC1 knockdown parasites became more resistant to M-512 suggesting that the compound is an agonist of both proteins which may form a functional complex and that dysregulation of this complex is deleterious to parasite growth.

microbiology↗

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

The Dual Action of Human Antibodies Specific to Plasmodium falciparum PfRH5 and PfCyRPA: Blocking Invasion and Inactivating Extracellular Merozoites

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines. Author SummaryMalaria is a sometimes-fatal disease caused by protozoan parasites of which Plasmodium falciparum is the most deadly species that causes hundreds of millions of infections and half a million deaths per year. A partially effective vaccine is available to block parasite forms transmitted by mosquitoes but not the subsequent blood stage which causes symptomatic disease. To fight blood stage parasites, proteins have been identified such as PfRH5, that aid parasite entry into human red blood cells (RBCs) and vaccines made from these proteins can trigger the production of antibodies that bind the parasite proteins thereby blocking RBC invasion. PfRH5 forms a complex with another parasite protein called PfCyRPA and together antibodies to PfCyRPA and PfRH5 are highly effective in reducing parasite growth. Here we investigated how antibodies to PfCyRPA and PfRH5 actually block invasion using video microscopy of live parasites. As anticipated, we found the antibodies not only stopped most parasites from invading but of those parasites that did invade, they took longer to do so, suggesting the antibodies were physically inhibiting the invasion process. One unanticipated effect of both PfRH5 antibodies and one of three PfCyRPA antibodies tested, was that they triggered the uninvaded parasites to change into cellular forms normally only seen inside RBCs. These intracellular forms are no longer competent to invade and so the PfRH5/CyRPA antibodies have the potential of both neutralize parasites by physically preventing RBC entry and by changing the parasites into invasion incompetent forms.

microbiology↗