Malaria is a devastating disease caused by Plasmodium parasites. Plasmodium parasites express ten cathepsin D-like aspartyl proteases, called plasmepsins (PMs). These PMs have diverse roles fulfill diverse functions throughout the parasite's lifecycle, though several exhibit functional redundancies. Among them, PMV, PMIV, and PMX are essential for asexual stage development, positioning them as prime candidates for antimalarial development. This review synthesizes current knowledge on PM biology and highlights the pivotal role of PM inhibitors in validating these proteases as therapeutic targets. Learnings from past research on PMs coupled with advances in experimental approaches have largely steered drug discovery efforts toward the development of dual PMIX/PMX inhibitors. Highlighted is an overview of the developmental trajectory of PM inhibitors, their progress to date, and their potential integration into future antimalarial therapies aimed at both prevention and treatment.

Parasitic helminth infections impose major burdens on human and animal health, and increasing anthelmintic resistance reinforces the need for new drugs. Early-stage discovery remains constrained by the scarcity of novel, potent, selective and chemically tractable starting points, the limited predictive value of some experimental models, mechanistic uncertainty and insufficient capacity to sustain promising series through medicinal chemistry, safety and exposure studies. Discovery can begin through complementary routes, including target-agnostic whole-organism screening, screening of known-pharmacology and repurposing collections, target- or pathway-led approaches and structure-guided or computational selection. Whole-organism models are valuable because they capture compound access and integrated parasite responses, but do not establish mechanism; conversely, target-based approaches require validation in the intact parasite. This review presents an iterative framework, connecting these entry routes with chemical diversity, hit confirmation and triage, early structure-activity relationship analysis, fit-for-purpose mechanistic investigation and translational assessment. Nematode models provide scalable platforms, while selected trematode and cestode examples illustrate broader applicability. Chemoproteomics, functional genomics and multi-omics can link phenotypes to candidate targets and pathways, while artificial intelligence can support compound and hypothesis prioritisation. Their value lies in informing experimental decisions rather than replacing validation. Quantitative examples show that measurable activity can be identified, but development-quality hit series remain rare. Scientific integration is therefore necessary but insufficient: sustained investment, cross-sector expertise, development infrastructure and continuity of project ownership are also required. Together, these principles define a balanced, experimentally grounded and mechanism-informed approach to early-stage anthelmintic discovery.

Intrinsic apoptosis is a form of programmed cell death that underpins development, tissue homeostasis and stress responses across Metazoa. In roundworms (nematodes), the pathway was first genetically defined in the free-living nematode Caenorhabditis elegans, yet how it has diversified and operates across the phylum Nematoda, encompassing parasites of humans and animals spanning clades I-V, remains incompletely resolved. Here, we synthesise comparative genomic, structural and functional evidence to establish a framework for intrinsic apoptosis in nematodes. Although the core CED-9-CED-4-CED-3 module is broadly retained, regulatory wiring and developmental deployment remain largely uncharacterised beyond C. elegans. Unlike vertebrates, nematodes lack a canonical BAX/BAK-driven mitochondrial permeabilisation system, revealing what we term the "Nematode Apoptosis Paradox" - caspase activation in the absence of the mitochondrial amplification step central to vertebrate intrinsic apoptosis. This alternative regulatory configuration, coupled with structural divergence of nematode BCL-2-like proteins from their vertebrate homologues, suggests a distinctive evolutionary trajectory for apoptotic regulation in Nematoda. By integrating evolutionary cell biology with emerging structural and pharmacological insights, we define a conceptual framework for interrogating apoptosis across clades I-V and evaluate its potential as a target for anthelmintic discovery.

ABSTRACT Plasmodium falciparum invasion of human erythrocytes is a complex and tightly coordinated process, involving host cell attachment, moving junction formation and engagement of the parasite’s actomyosin motor. The temporal precision of these events is mediated by distinct ligand-receptor interactions and the sequential release of the merozoite’s 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 Ca 2+ flux but prevents host cytoskeleton disassembly. In contrast, the inhibition of parasite actin polymerisation had no effect on cytoskeletal clearance but caused a sustained Ca 2+ 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.

Abstract Plasmepsin V is an essential aspartyl protease that cleaves the Plasmodium export element in proteins targeted for export from Plasmodium parasites into the host red blood cell. Substrate-mimicking peptidomimetics have been developed that potently inhibit plasmepsin V but show modest parasite activity. To advance these inhibitors, we optimized their potency by introducing a 2-chloro aryl group at P2 and an N-terminal sulfonamide that yielded WM960, which showed markedly improved plasmepsin V inhibition while retaining high selectivity over plasmepsin IX and X and human aspartyl proteases. WM960 also demonstrated enhanced activity against asexual blood-stage parasites and effectively suppressed protein export. However, WM960 displayed low metabolic stability and limited systemic exposure in mice, highlighting the need to improve pharmacokinetic properties in future development of peptidomimetic plasmepsin V targeted antimalarials.

The control of parasitic nematodes of humans and animals remains heavily dependent on a limited number of anthelmintic drug classes, and resistance to major classes is now widespread. Although phenotypic screening readily identifies compounds that impair worm motility or development, the intrinsic biological processes underlying chemical sensitivity in parasitic nematodes remain poorly defined. Here, we identified a hit compound with a pyridyl scaffold from a phenotypic screen against the model parasitic nematode, Haemonchus contortus, and using structure–activity optimisation we generated a potent chemical probe, WEHI-684. To uncover protein networks associated with the mechanism of action, thermal proteome profiling and time-resolved quantitative proteomics interrogated WEHI-684-induced perturbations in H. contortus. Across larval and adult stages of this major parasite of livestock, proteome integral solubility alteration (PISA) profiling revealed reproducible alterations in proteins associated with cytoskeletal organisation and intracellular trafficking, including actin- and motor-related components. Complementary quantitative proteomics identified induction of an aspartyl protease and suppression of secretory CAP family proteins. Integrated analysis of these datasets supports a model in which chemical perturbation of cytoskeletal and trafficking proteins is associated with secondary modulation of proteolytic pathways, coinciding with rapid impairment of motility. These findings indicate that linked structural and proteolytic responses contribute to chemical sensitivity in H. contortus and demonstrate how integrative proteomics can resolve organism-level responses to chemical perturbation beyond single-target paradigms.

With resistance to current frontline antimalarial treatments rapidly emerging in malaria endemic regions, there is an urgent need to identify new antimalarial compounds with novel mechanisms of action. Currently, no clinically used antimalarials target erythrocyte invasion, the process by which the short-lived, extracellular merozoite form of Plasmodium falciparum parasites enters a host erythrocyte. Following invasion, the parasite grows and then divides to form a schizont, containing merozoites that exit the host cell and invade new erythrocytes. To identify novel egress and invasion inhibitors, we screened the Medicines for Malaria Venture COVID Box (160 compounds) and Global Health Priority Box (240 compounds). Parasites were treated with the compounds at 1 μM during the egress and invasion period and across both libraries, 20 invasion inhibitors and two partial egress inhibitors were identified. A comparison of the growth IC50 and invasion IC50 of each hit compound revealed that invasion inhibition is likely central to the parasiticidal mechanism of six of the compounds, as their IC50s for growth and invasion were similar. Of these six inhibitors, two (MMV006931 and MMV024850) were found to directly block the invasion of mechanically purified merozoites. We sought to identify the protein targets of these compounds through resistance selection and subsequent whole genome sequencing. We were able to select parasites with robust resistance to MMV006931 but not MMV024850. Genome sequencing of clonal MMV006931-resistant parasites identified mutations in the P. falciparum sterol exporter PfNCR1, which helps eliminate excess cholesterol from the parasite's plasma membrane. By demonstrating that MMV006931 sensitises parasites to lysis by a sterol-dependent detergent, we confirmed that PfNCR1 is the likely target of MMV006931.

The emergence of resistance to most clinically used antimalarials necessitates the discovery of new chemotypes. A phenotypic screen against asexual Plasmodium falciparum identified the 2-amino-3,4-dihydroquinazoline scaffold, previously developed as a β-secretase 1 inhibitor for Alzheimer's disease. Evaluation of hit analogues against malarial aspartyl proteases revealed potent inhibition of plasmepsin X. Structure-activity analysis showed that key motifs on the hit scaffold are required for driving biochemical and antimalarial but are associated with poor selectivity against human aspartyl proteases and low metabolic stability. Plasmepsin X was validated as the molecular target through forward genetics, activity against mutant parasites, and inhibition of plasmepsin X substrate processing. 2-Amino-3,4-dihydroquinazoline analogs exhibit moderate asexual killing rates, low-to-moderate resistance risk, no cross-resistance with multidrug-resistant strains, and transmission-blocking activity. Further optimization of potency and metabolic stability will be required to achieve in vivo efficacy and realize the potential of this antimalarial class.

BackgroundProgrammed cell death (or apoptosis) is a fundamental process in metazoans, extensively characterised in mammals and other vertebrates but much less so in invertebrates beyond the free-living nematode Caenorhabditis elegans and the vinegar fly - Drosophila melanogaster. Here, we present the first reconstruction of the complete intrinsic apoptosis pathway in the parasitic nematode Haemonchus contortus, a blood-feeding pathogen of ruminants and a major cause of global production losses.ResultsUsing C. elegans proteins as references, we combined genome-wide homology searches, structural modelling, and developmental transcriptomic and proteomic analysis to identify and characterise apoptosis regulators in H. contortus. Homologues of all canonical C. elegans components were found, including CEP-1, EGL-1, CED-9, CED-4 and CED-3, together with modulators such as DRE-1 and PUF-8. Structural models revealed conservation of the CED-9:CED-4 and CED-4:CED-3 complexes, while EGL-1 and CEP-1 retained key structural domains despite significant sequence divergence. Transcriptomic data showed that the genes Hc-ced-9 and Hc-ced-3 are constitutively expressed across developmental stages, whereas Hc-cep-1 and Hc-egl-1 display stage-specific transcription. Proteomic data confirmed the presence of Hc-CED-9, Hc-CED-4 and Hc-CED-3 in at least one developmental stage, while Hc-EGL-1 and Hc-DRE-1 were not detected. Discordances between RNA and protein profiles, particularly for Hc-EGL-1, suggest tight post-transcriptional control. These findings demonstrate that, while the core architecture of apoptosis is conserved in H. contortus, regulatory divergence has occurred, reflecting lineage-specific adaptations.ConclusionThis molecular framework highlights conserved structural features and developmental regulation of apoptosis in a parasitic nematode and provides a basis for functional studies to evaluate apoptotic regulators as potential targets for anthelmintic development.

The emergence of resistance to most clinically used antimalarials necessitates the discovery of new chemotypes. A phenotypic screen against asexual identified the 2-amino-3,4-dihydroquinazoline scaffold, previously developed as a beta-secretase 1 inhibitor for Alzheimer's disease. Evaluation of hit analogues against malarial aspartyl proteases revealed potent inhibition of plasmepsin X. Structure-activity analysis showed that key motifs on the hit scaffold are required for driving biochemical and antimalarial but are associated with poor selectivity against human aspartyl proteases and low metabolic stability. Plasmepsin X was validated as the molecular target through forward genetics, activity against mutant parasites, and inhibition of plasmepsin X substrate processing. 2-Amino-3,4-dihydroquinazoline analogs exhibit moderate asexual killing rates, low-to-moderate resistance risk, no cross-resistance with multidrug-resistant strains, and transmission-blocking activity. Further optimization of potency and metabolic stability will be required to achieve in vivo efficacy and realize the potential of this antimalarial class.

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, but emerging resistance threatens their efficacy. The potential for the development of drug resistance against plasmepsin X (PMX)-selective inhibitors and dual plasmepsin IX/X (PMIX/X) inhibitors was investigated in Plasmodium falciparum. A series of PMX-selective (WM4, WM76, WM92) and PMIX/X dual inhibitors (WM382, WM09, WM42) were characterised for potency against parasite growth and enzyme inhibition. In vitro selection experiments showed that all compounds had a high barrier to resistance, although parasites with reduced sensitivity to PMX‑selective inhibitors could still be selected. Resistance mechanisms involved pmx gene amplification and point mutations (D245N, S315P, S359P, I363L) that alter inhibitor binding. Recombinant expression and Michaelis-Menten kinetics demonstrated that these mutations impair drug binding whilst preserving PMX catalytic function. Reverse genetics confirmed that introducing these mutations into the pmx gene resulted in decreased potency of the inhibitors. In this study, resistance to the PMIX/X dual inhibitors evaluated here could not be selected, despite prolonged selection pressure. Antimalarial Resistome Barcoding (AReBar) assays confirmed the absence of pre-existing resistance to either inhibitor class. Critically, PMIX/X dual inhibitors maintained efficacy against parasites with decreased sensitivity to PMX-selective compounds. These findings demonstrate that dual PMIX/X inhibitors present a substantially higher barrier to resistance than PMX-selective inhibitors, informing antimalarial drug development strategies and highlighting dual-target inhibition as a promising approach to mitigate resistance risks.

Background The global burden of malaria remains substantial, and increasing parasite resistance to current antimalarials necessitates the development of drugs with unique mechanisms of action. This study aimed to develop and characterise a new antimalarial compound targeting Plasmodium aspartic proteases. Methods We conducted high-throughput screening, medicinal chemistry optimisation, and extensive in vitro and in vivo testing to develop and evaluate MK-7602, a dual inhibitor of plasmepsins IX and X. Findings MK-7602, a clinical candidate, acts as a dual sub-nanomolar inhibitor of plasmepsins IX and X in multiple Plasmodium species. It exhibits favourable pharmacokinetic properties and a promising safety profile. MK-7602 demonstrates activity against liver and blood life-cycle stages of the parasite and blocks transmission to mosquitoes. Importantly, it shows a high barrier to resistance development and lacks cross-resistance with Plasmodium falciparum strains resistant to other antimalarials. MK-7602 effectively inhibits both wild-type parasites and those with increased plasmepsin expression, highlighting its potential to overcome existing resistance mechanisms. Interpretation MK-7602 represents a new class of antimalarial for treating uncomplicated malaria with a new mechanism of action and the potential to address drug-resistant malaria. Clinical evaluation of MK-7602's activity against P. falciparum is ongoing. Funding This work was funded by The Wellcome Trust (109662/Z/15/Z, 202749/Z/16/Z, 219658/Z/19/Z), NHMRC (GNT1176955, GNT637406, GNT1173049), the Human Frontiers Science Program (LT0001/2022-L, JMD), Drakensberg Trust, the Victorian State Government Operational Infrastructure Support grant, and the Australian Government NHMRC IRIISS. JPo was supported by the NIH/NIAID (R01AI173171, R01AI175134 and R61AI187100) and the Pasteur International Unit PvESMEE.

New antimalarials are needed due to the threat of emerging resistance against existing antimalarial therapies. A phenotypic screen uncovered the N-aryl acetamide class that inhibits the development of P. falciparum asexual ring-stage parasites. The structure-activity relationship of this class was investigated, and key modifications were introduced that produced WEHI-326 with potent antimalarial activity. Enhancing the metabolic stability of this class will be a future challenge to achieve efficacy in a malaria mouse model. WEHI-326 was found to have a moderate barrier to resistance and a moderate rate of asexual kill, potently inhibited gametocyte and gamete development, and in turn, blocked the transmission of parasites to the mosquito. Forward genetics and cross-resistance profiling determined that parasites resistant to N-aryl acetamides had mutations in rhomboid protease 8 (ROM8) and the putative cation channel, CSC1. WEHI-326 will be an important tool in unraveling the role of ROM8 and CSC1 in P. falciparum development.

The spread of drug-resistant Plasmodium strains is diminishing the effectiveness of current antimalarials, highlighting the importance of discovering new therapeutics with novel targets. A screen of the Jumpstarter library against P. falciparum identified W482 with a pyrimidine-2,4-diamine scaffold. Structure-activity relationships reveal the importance of the pyrimidine core and its endocyclic nitrogen, while alternative amines are tolerated in the 4-position. Bulky and hydrophobic carboxamides or substituted phenyl ureas display the most potent antiplasmodial activity. Resistance selection and whole genome sequencing reveal an amplification of the gene encoding the ABCI3 transporter protein W482-resistant parasites. W482 is found to exhibit greater activity against parasites with reduced expression of ABCI3, confirming that resistance is related to the transporter. W482 arrests asexual parasites at the ring to trophozoite transition stage and exhibits a fast-killing profile with a lag phase of 24 h. Improving the antiparasitic activity alongside metabolic stability and solubility remains a challenge in the future development of the pyrimidine-2,4-diamine class.

Parasitic nematodes pose a significant threat to human and animal health, causing widespread morbidity and substantial socioeconomic losses globally. Despite the utility of anthelmintic drugs in parasite control, the emergence of widespread resistance necessitates the discovery of novel interventions. Advances through the use of whole-organism phenotypic screening have identified some promising nematocidal compounds, including nemacol, tolfenpyrad, UMW-9729, and ABX464. This article summarises efforts in this discovery, with a focus on Haemonchus contortus and Caenorhabditis elegans as model nematodes, and discusses approaches used for drug target deconvolution, including proteomic, chemical and genetic/genomic techniques. Stability-based proteomic assays, such as thermal proteome profiling, have been useful for identifying protein targets for these compounds, shedding light on their mechanisms of action. However, challenges remain in extrapolating findings from C. elegans to parasitic nematodes, emphasising the need for validation studies. Understanding drug–target interactions in nematodes is critical for developing next-generation anthelmintics and for mitigating the growing resistance challenge. This review outlines recent progress in this area and discusses future directions in target validation and anthelmintic development to support parasite control programmes.

Drug resistance is steadily undermining the efficacy of frontline anti-malarials, highlighting the urgent need for novel therapies with alternative mechanisms of action. The chemical addition of different moieties to azithromycin yields compounds with improved quick-killing potency against malaria parasites, with the most active analogs typically containing a chloroquinoline group. Here, we investigated the quick-killing mechanism of five azithromycin analogs, two of which contain differentially oriented chloroquinoline moieties. The improvement in quick-killing activity over azithromycin for non-chloroquinoline analogs was around 10 -to 42-fold, with chloroquinoline-containing analogs showing a further 2- to 17-fold improvement over non-chloroquinoline compounds. Chemical inhibition of hemoglobin digestion and chloroquine's inhibitory effect against heme polymerization linked analogs with both chloroquinoline and non-chloroquinoline modifications to a chloroquine-like mechanism of action. However, none of the analogs showed a significant reduction in efficacy against chloroquine-resistant asexual blood-stage parasites. Multiple attempts at selecting for azithromycin analog-resistant parasites to elucidate the mechanism of quick-killing were unsuccessful. Application of cellular thermal shift proteomics revealed that azithromycin analogs significantly stabilized 34-155 different proteins in trophozoites, a high number that showed minimal overlap with chloroquine. Additionally, our most potent chloroquinoline-containing analog demonstrated a significant improvement in gametocytocidal activity over azithromycin and further maintained moderate inhibition of chloroquine-insensitive late-stage gametocytes. These findings support that this class of azithromycin analogs kills malaria parasites through a broad range of potential mechanisms, making them promising candidates for optimization as fast and broad-acting anti-malarials.

To discover new antimalarials, a screen of the Janssen Jumpstarter library against Plasmodium falciparum uncovered the N-acetamide indole hit class. The structure-activity relationship of this chemotype was defined and culminated in the optimized frontrunner analog WJM664, which exhibited potent asexual stage activity and high metabolic stability. Resistant selection and whole-genome sequencing revealed mutations in PfATP4, which was validated as the target by showing that analogs exhibited reduced potency against parasites with resistance-conferring mutations in PfATP4, a metabolomic signature similar to that of the PfATP4 inhibitor KAE609, and inhibition of Na+-dependent ATPase activity consistent with on-target inhibition of PfATP4. WJM664 inhibited gamete development and blocked parasite transmission to mosquitoes but exhibited low efficacy in aPlasmodium berghei mouse model, which was attributed to ATP4 species differentiation and its moderate systemic exposure. Optimization of these attributes is required for N-acetamide indoles to be pursued for development as a curative and transmission-blocking therapy.

Despite the substantial global health and economic burden of apicomplexan parasites in humans and livestock, treatment options remain limited. Natural products have long played an important role in combating these diseases, offering diverse chemical structures and bioactive compounds. This review summarises past and present natural-product-based therapies for six economically significant apicomplexans and explores the potential of revisiting natural products as a source of next-generation treatments.

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 and intact-cell thermal 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 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.

A library of known aspartic protease inhibitors was screened to identify compounds that inhibit plasmepsin V from Plasmodium vivax. This screen revealed compounds with an imino-pyrimidinone-fused pyrrolidine (IPF) scaffold that exhibited sub-micromolar inhibitory activity against plasmepsin V. Further screening of IPF analogs against the related aspartic protease plasmepsin X showed inhibitory activity, while a third aspartic protease, plasmepsin IX, was not significantly inhibited. Modifications to the P1 biaryl region of the IPF scaffold differentially modulated inhibition of both plasmepsin V and X. Notably, analogs with potent plasmepsin X inhibitory activity successfully blocked the growth of Plasmodium falciparum in vitro. X-ray structures of IPF analogs in complex with plasmepsin V provided insights into their binding mode and revealed avenues to further improve IPF potency and selectivity between plasmepsin V and X. This understanding of how these compounds interact with the active sites of plasmepsin V and X will serve as a foundation for the future design of dual inhibitors targeting these proteases.