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.

Fasciola hepatica causes fasciolosis in livestock and humans worldwide, yet reliable tools to assess drug efficacy against the early developmental stages of this parasite are lacking. Here, we developed an automated miracidial motility assay (MMA) using the WMicroTracker ONE infrared detection system to quantify the sensitivity of F. hepatica miracidia to anthelmintic compounds including clorsulon (CLORS), closantel (CLOS), triclabendazole (TCBZ) and triclabendazole-sulphoxide (TCBZ-SO). Systematic optimisation of assay conditions, including inoculum size, observation window and solvent concentration yielded a reliable platform for evaluating the sensitivity of F. hepatica miracidia from diverse geographic isolates to these compounds. Our results demonstrated that three compounds (CLOS, TCBZ and TCBZ-SO) produced concentration-dependent motility inhibition, whereas CLORS had no effect. CLOS displayed the highest potency among isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC), whereas TCBZ and TCBZ-SO exhibited isolate-specific sensitivity patterns. Miracidial responses of the NSW and TAS isolates to TCBZ, TCBZ-SO and CLOS were also compared in vitro with those of newly excysted juveniles (NEJs) produced from the same isolates. Overall, the findings show that MMA provides a reproducible, host-independent and high-throughput phenotypic platform for assessing miracidial sensitivity to compounds.

Parasitic helminths impose substantial health and economic burdens on humans and livestock, and widespread resistance to existing anthelmintic classes underscores the urgent need for new chemotypes with distinct mechanisms of action as part of integrated parasite control strategies. Here, we performed a large-scale phenotypic screen of the Medicines for Malaria Venture Hit Generation Library 1 (HGL1), testing 139,916 compounds (98.7% of the 141,786-compound library) against exsheathed third-stage larvae of Haemonchus contortus, with selective cross-species evaluation in Caenorhabditis elegans. Using infrared-based motility and developmental assays in 384-well format, the platform delivered excellent performance across > 360 plates (mean Z′ = 0.799 ± 0.012; signal-to-background = 65.6 ± 9.8). We identified 272 primary hits (0.194%) and confirmed 110 active compounds with reproducible inhibition of larval motility and development. Of these, 39 exhibited IC50 < 10 μM and 33 caused complete developmental arrest at ≤ 12.5 μM, accompanied by characteristic phenotypes such as eviscerated, curved and coiled forms. Four of the 39 compounds were non-toxic to HepG2 cells (CC50 ≥ 20 μM; MC50 ≥ 20 μM), and a subset displayed favourable physicochemical properties (logD < 3; polar surface area < 100 Å2; metabolic stability > 60%), with ADME (absorption, distribution, metabolism and excretion) profiling available for a prioritised subset. Integration of potency, selectivity and/or ADME data enabled the prioritisation of candidates, including 16 that met stringent criteria for medicinal-chemistry progression. These findings demonstrate that a chemically curated library originally developed for antimalarial discovery might yield potent, selective nematocidal scaffolds and support a scalable framework for repurposing discovery libraries across divergent parasite groups.

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.

About 50 years after their development, the benzimidazole anthelmintic drugs still occupy a key role in the treatment of parasitic diseases. Like other anti-infective drugs, their extensive use has led to resistance development and now requires new solutions to treat these diseases. Organometallic derivatization of existing drugs has become a common strategy to design new drug candidates, which could benefit from enhanced activity against resistant or sensitive strains through the incorporation of an organometallic fragment. In this study, we describe the development of a new series of organometallic derivatives of benzimidazole anthelmintics. Ten derivatives, six of which were organometallic, were synthesized and characterized. In silico and in vitro studies were performed to determine their biological activity. Therefore, they were screened against a range of nematodes and other parasites. Biological activities were found for some of the derivatives against Strongyloides ratti, Ancylostoma ceylanicum, Trichuris muris, Schistosoma mansoni, Echinococcus multilocularis, or Leishmania infantum in similar ranges as the parent anthelmintics. Even if the activity of the benzimidazole scaffold was not systematically enhanced, this strategy is a good starting point for the development of new antiparasitic compounds and for further study of the mode of action of these drugs.

Gastrointestinal nematodes, including Haemonchus contortus, represent a major constraint to livestock production globally, and the increasing prevalence of anthelmintic resistance necessitates the discovery of new chemotypes. Natural products provide a rich and underexplored source of bioactive scaffolds for anthelmintic development. However, there remains a need to systematically optimise such scaffolds to define structure-activity relationships in whole-organism systems. Here, we investigated neolignan compounds from the fruits of Styrax suberifolius and assessed the impact of halogenation on their anthelmintic activity. Two known neolignans, suberifolioside A and equiselignan B, were isolated and used to generate a series of 11 halogenated derivatives using N-halosuccinimide chemistry. In addition, a new glycoside, suberifolioside B, together with two known metabolites, 7R,8S-dihydrodehydrodiconiferyl alcohol and 3',4-O-dimethylcedrusin, were characterised by spectroscopic and spectrometric methods, and the absolute configuration of equiselignan B was confirmed by X-ray crystallography. All compounds were evaluated for activity against larval stages of H. contortus using an established in vitro phenotypic assay. While no compounds significantly affected exsheathed third-stage larvae, multiple derivatives displayed activity against fourth-stage larvae, indicating pronounced stage-specific susceptibility. Notably, halogenation of the neolignan scaffold modulated biological activity, with several derivatives inducing substantial reductions in larval motility and distinct abnormal phenotypes. This modulation was non-linear, with both mono- and poly-halogenated derivatives displaying activity. A monobrominated ether analogue exhibited maximal motility inhibition approaching that observed for moxidectin under the assay conditions, highlighting the potential of this scaffold for optimisation. These findings demonstrate that halogenated neolignans represent a tractable chemical class for anthelmintic discovery and reveal stage-specific vulnerabilities in H. contortus. This work provides a foundation for further optimisation and prioritisation of neolignan derivatives for downstream studies of mechanism of action and translational potential.

Parasitic nematodes cause a wide range of diseases in animals, including humans. However, the efficacy of existing anthelmintic drugs, commonly used to treat these infections, is waning due to the increasing prevalence of drug resistance in nematode populations. This growing challenge underscores the urgent need to discover and develop novel nematocidal drugs that target new molecular pathways. In the present study, 13 novel derivatives of benzhydroxamic acid (OMKs) were designed and synthesized. Their anthelmintic activity was tested in the parasitic nematode Haemonchus contortus (barber's pole worm) and the free-living nematode Caenorhabditis elegans and potential toxicity assessed in mammalian models. Compound OMK211 showed the most promising results. It decreased viability and motility of larval and adult stages of both nematode species and of both drug-sensitive and drug-resistant strains of H. contortus at micromolar concentrations with the highest efficacy in H. contortus adult males (IC50 ∼ 1 μM). Moreover, OMK211 was not toxic in mammalians cells in vitro and in mice in vivo. Consequently, thermal proteome profiling analysis was used to infer the putative molecular target of OMK211 in H. contortus. The results revealed C2-domain containing protein A0A6F7Q0A8, encoded by gene HCON_00184,900, as an interacting partner of OMK211. Using advanced structural prediction and docking tools, this protein is considered an interesting putative molecular target of new nematocidal drugs as its orthologs are present in several nematodes but not in mammals. In conclusion, novel derivatives of benzhydroxamic acid represent a promising new class of potential anthelmintics, which deserve further testing.

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.

A high-throughput platform for assessing the activity of synthetic or natural compounds on the motility and development of Haemonchus contortus larvae has been established for identifying new anthelmintic compounds active against strongylid nematodes. This study evaluated the impact of serum supplementation on larval development, motility and survival in vitro and its implications for phenotypic compound screening. Of five blood components assessed, 7.5% sheep serum significantly enhanced larval development, motility and survival compared to the original medium (LB*), leading to the formulation of an improved medium (LBS*). Proteomic analysis revealed marked differences in protein expression in larvae cultured in LBS* versus LB*, including molecules associated with structural integrity and metabolic processes. The phenotypic screening of 240 compounds (“Global Priority Box” from Medicines Malaria Venture) using LBS* yielded results distinct from those in LB*, highlighting the effect of culture conditions on screening assessments. These findings indicate/emphasise the critical need to evaluate and optimise culture media for physiologically relevant conditions in screening platforms, improving the reliability of anthelmintic discovery.

A recent high-throughput screening of the NatureBank marine extract library (7616 samples) identified an extract from the Australian marine sponge Agelas axifera with in vitro activity against an economically important parasitic nematode, Haemonchus contortus (barber’s pole worm). The bioassay-guided fractionation of the CH2Cl2/MeOH extract from A. axifera led to the purification of a new diterpene alkaloid, agelasine Z (1), together with two known compounds agelasine B (2) and oxoagelasine B (3). Brominated compounds (–)-mukanadin C (4) and 4-bromopyrrole-2-carboxylic acid (5) were also isolated from neighbouring UV-active fractions. All compounds, together with agelasine D (6) from NatureBank’s pure compound library, were tested for in vitro anthelmintic activity against exsheathed third-stage (xL3s) and fourth-stage larvae (L4s) of H. contortus and young adult Caenorhabditis elegans. Compounds 1, 2 and 6 induced an abnormal “skinny” phenotype, while compounds 2 and 6 also reduced the motility of H. contortus L4s by 50.5% and 51.8% at 100 µM, respectively. The minimal activity of agelasines against C. elegans young adults suggests a possible species-specific mechanism warranting further investigation. For the first time, the unexpected lability of agelasine H-8′ was explored using kinetic studies, revealing rapid deuterium exchange in MeOH-d4 at room temperature.

The plant metabolite, equiselignan B (2), isolated from the fruits of Styrax suberifolius was used to generate a semisynthetic library. Extraction and purification studies yielded large quantities (similar to 70 mg) of 2, a new glycoside, suberifolioside A (1), and the previously described neolignan egonol (3). The planer structure of 1 was established following 1D/2D NMR and MS analyses, and its absolute configuration was determined by X-ray diffraction studies. Acid hydrolysis of 1 yielded an additional amount of scaffold 2 (436 mg) that was subsequently converted into seven new ether derivatives (4-10) and two new ester derivatives (11-12) using commercially available alkyl halides and acyl chlorides, respectively. The chemical structures of the new semisynthetic ether and ester derivatives were assigned by spectroscopic and spectrometric analyses. Subsequently, compounds 1-12 were evaluated for their anthelmintic activity on exsheathed third-stage larvae (xL3s) and fourth-stage larvae (L4s) of Haemonchus contortus (barber's pole worm) - a highly pathogenic parasitic roundworm of ruminant livestock. Ether derivative 5 displayed significant anthelmintic activity against both xL3s and L4s, resulting in a 66-73 % reduction in motility at 50 mu M after 168 h (xL3s) or 90 h (L4s) of exposure and IC50 values of 24-30 mu M. This derivative also induced curved (Cur), evisceration (Evi) and skinny (Ski) phenotypes in affected larvae. Compounds 1, 9 and 12 also had marked activity against L4s, which indicate that the neolignan structure class warrants further investigation for anthelmintic activity.

The control of socioeconomically important parasitic roundworms (nematodes) of animals has become challenging or ineffective due to problems associated with widespread resistance in these worms to most classes of chemotherapeutic drugs (anthelmintics) currently available. Thus, there is an urgent need to discover and develop novel compounds with unique mechanisms of action to underpin effective parasite control programmes. Here, we evaluated an in silico (computational) approach to accelerate the discovery of new anthelmintics against the parasitic nematode Haemonchus contortus (barber’s pole worm) as a model system. Using a supervised machine learning workflow, we trained and assessed a multi-layer perceptron classifier on a labelled dataset of 15,000 small-molecule compounds, for which extensive bioactivity data were previously obtained for H. contortus via high-throughput screening, as well as evidence-based datasets from the peer-reviewed literature. This model achieved 83% precision and 81% recall on the class of ‘active’ compounds during testing, despite a high imbalance in the training data, with only 1% of compounds carrying this label. The trained model was then used to infer nematocidal candidates by in silico screening of 14.2 million compounds from the ZINC15 database. An experimental assessment of 10 of these candidates showed significant inhibitory effects on the motility and development of H. contortus larvae and adults in vitro, with two compounds exhibiting high potency for further exploration as lead candidates. These findings indicate that the present machine learning-based approach could accelerate the in silico prediction and prioritisation of anthelmintic small molecules for subsequent in vitro and in vivo validations.

Australian plants of the family Haemodoraceae have been a reliable source of new secondary metabolites, particularly those of the ‘phenylphenalenone’ class, and related chromenes and xanthones. Some of these compounds demonstrate anti-microbial properties against both Gram-negative and Gram-positive bacteria. Chemical profiling of thirty individual ethanolic extracts from six separate species of Australian plants belonging to the family Haemodoraceae was conducted using an HPLC-MS approach reinforced by HRLC(ESI)-MS. Six of the extracts were further explored by employing HRLC(ESI)-MS and the compounds present were characterised and confirmed based on a comparison to the original data. All thirty extracts were assessed for biological activity against the parasitic nematode Haemonchus contortus in vitro. The chemical profiling methodology adopted resulted in the identification of thirty-four previously reported compounds, identifying on average 64% of the previously reported secondary metabolites across the species Haemodorum simulans, Haemodorum spicatum, Haemodorum brevisepalum and Macropidia fuliginosa. Furthermore, compounds from the phenylbenzoisoquinolindone class were detected in the bulbs of Haemodorum simulans and Haemodorum coccineum, representing the first report of the structure class in extracts of the genus Haemodorum. Extracts of the H. simulans stems, M. fuliginosa bulbs and H. distichophyllum roots and bulbs exhibited anthelmintic activity in vitro. The chemical profiling HPLC-MS methodology adopted was successful in the rapid identification of most of the previously reported secondary metabolites across the Haemodoracae species, indicating that the analytical approach was robust. This study demonstrates the effectiveness of dereplication via HPLC-MS-based chemical profiling across six Australian Haemodoraceae species, identifying numerous known and putatively novel secondary metabolites. It also reports, for the first time, anthelmintic activity in selected species and marks the first detailed phytochemical investigation of H. distichophyllum since its initial pigment analysis over 50 years ago.

Advances in single cell technologies are allowing investigations of a wide range of biological processes and pathways in animals, such as the multicellular model organism Caenorhabditis elegans – a free-living nematode. However, there has been limited application of such technology to related parasitic nematodes which cause major diseases of humans and animals worldwide. With no vaccines against the vast majority of parasitic nematodes and treatment failures due to drug resistance or inefficacy, new intervention targets are urgently needed, preferably informed by a deep understanding of these nematodes’ cellular and molecular biology – which is presently lacking for most worms. Here, we created the first single cell atlas for an early developmental stage of Haemonchus contortus – a highly pathogenic, C. elegans-related parasitic nematode. We obtained and curated RNA sequence (snRNA-seq) data from single nuclei from embryonating eggs of H. contortus (150,000 droplets), and selected high-quality transcriptomic data for > 14,000 single nuclei for analysis, and identified 19 distinct clusters of cells. Guided by comparative analyses with C. elegans, we were able to reproducibly assign seven cell clusters to body wall muscle, hypodermis, neuronal, intestinal or seam cells, and identified eight genes that were transcribed in all cell clusters/types, three of which were inferred to be essential in H. contortus. Two of these genes (i.e. Hc-eef-1A and Hc-eef1G), coding for eukaryotic elongation factors (called Hc-eEF1A and Hc-eEF1G), were also demonstrated to be transcribed and expressed in all key developmental stages of H. contortus. Together with these findings, sequence- and structure-based comparative analyses indicated the potential of Hc-eEF1A and/or Hc-eEF1G as intervention targets within the protein biosynthesis machinery of H. contortus. Future work will focus on single cell studies of all key developmental stages and tissues of H. contortus, and on evaluating the suitability of the two elongation factor proteins as drug targets in H. contortus and related nematodes, with a view to finding new nematocidal drug candidates.

Millions of livestock animals worldwide are infected with the haematophagous barber’s pole worm, Haemonchus contortus, the aetiological agent of haemonchosis. Despite the major significance of this parasite worldwide and its widespread resistance to current treatments, the lack of a high-quality genome for the well-defined strain of this parasite from Australia, called Haecon-5, has constrained research in a number of areas including host-parasite interactions, drug discovery and population genetics. To enable research in these areas, we report here a chromosome-contiguous genome (∼280 Mb) for Haecon-5 with high-quality models for 19,234 protein-coding genes. Comparative genomic analyses show significant genomic similarity (synteny) with a UK strain of H. contortus, called MHco3(ISE).N1 (abbreviated as “ISE”), but we also discover marked differences in genomic structure/gene arrangements, distribution of nucleotide variability (single nucleotide polymorphisms (SNPs) and indels) and orthology between Haecon-5 and ISE. We used the genome and extensive transcriptomic resources for Haecon-5 to predict a subset of essential single-copy genes employing a “cross-species” machine learning (ML) approach using a range of features from nucleotide/protein sequences, protein orthology, subcellular localisation, single-cell RNA-seq and/or histone methylation data available for the model organisms Caenorhabditis elegans and Drosophila melanogaster. From a set of 1,464 conserved single copy genes, transcribed in key life-cycle stages of H. contortus, we identified 232 genes whose homologs have critical functions in C. elegans and/or D. melanogaster, and prioritised 10 of them for further characterisation; nine of the 10 genes likely play roles in neurophysiological processes, germline, hypodermis and/or respiration, and one is an unknown (orphan) gene for which no detailed functional information exists. Future studies of these genes/gene products are warranted to elucidate their roles in parasite biology, host-parasite interplay and/or disease. Clearly, the present Haecon-5 reference genome and associated resources now underpin a broad range of fundamental investigations of H. contortus and could assist in accelerating the discovery of novel intervention targets and drug candidates to combat haemonchosis.

Nanoparticles show great promise as a platform for developing vaccines for the prevention of infectious disease. We have been investigating a method whereby nanocapsules can be formulated from protein, such that the final capsules contain only the cross-linked protein itself. Such nanocapsules are made using a silica templating system and can be customised in terms of size and porosity. Here we compare the construction and characteristics of nanocapsules from four different proteins: one a model protein (ovalbumin) and three from infectious disease pathogens, namely the influenza virus, Helicobacter pylori and HIV. Two of the nanocapsules were assessed further. We confirm that nanocapsules constructed from the urease A subunit of H. pylori can reduce subsequent infection in a vaccinated mouse model. Further, we show that capsules constructed from the HIV gp120 protein can be taken up by dendritic cells in tissue culture and can be recognised by antibodies raised against the virus. These results point to the utility of this method in constructing protein-only nanocapsules from proteins of varying sizes and isoelectric points.

Abstract Background Infection with parasitic nematodes (helminths), particularly those of the order Strongylida (such as Haemonchus contortus), can cause significant and burdensome diseases in humans and animals. Widespread drug (anthelmintic) resistance in livestock parasites, the absence of vaccines against most of these nematodes, and a lack of new and effective chemical entities on the commercial market demands the discovery of new anthelmintics. In the present study, we searched the Global Health Priority Box (Medicines for Malaria Venture) for new candidates for anthelmintic development. Methods We employed a whole-organism, motility-based phenotypic screening assay to identify compounds from the Global Health Priority Box with activity against larvae of the model parasite H. contortus, and the free-living comparator nematode Caenorhabditis elegans. Hit compounds were further validated via dose–response assays, with lead candidates then assessed for nematocidal activity against H. contortus adult worms, and additionally, for cytotoxic and mitotoxic effects on human hepatoma (HepG2) cells. Results The primary screen against H. contortus and C. elegans revealed or reidentified 16 hit compounds; further validation established MMV1794206, otherwise known as ‘flufenerim’, as a significant inhibitor of H. contortus larval motility (half-maximal inhibitory concentration [IC50] = 18 μM) and development (IC50 = 1.2 μM), H. contortus adult female motility (100% after 12 h of incubation) and C. elegans larval motility (IC50 = 0.22 μM). Further testing on a mammalian cell line (human hepatoma HepG2 cells), however, identified flufenerim to be both cytotoxic (half-maximal cytotoxic concentration [CC50] < 0.7 μM) and mitotoxic (half-maximal mitotoxic concentration [MC50] < 0.7 μM). Conclusions The in vitro efficacy of MMV1794206 against the most pathogenic stages of H. contortus, as well as the free-living C. elegans, suggests the potential for development as a broad-spectrum anthelmintic compound; however, the high toxicity towards mammalian cells presents a significant hindrance. Further work should seek to establish the protein–drug interactions of MMV1794206 in a nematode model, to unravel the mechanism of action, in addition to an advanced structure–activity relationship investigation to optimise anthelmintic activity and eliminate mammalian cell toxicity. Graphical Abstract

Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25, that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation.

Tularemia is a deadly disease caused by Francisella tularensis, an emerging intracellular bacterial pathogen that can be disseminated rapidly through aerosols and vector-borne transmission. Recent surveillance data demonstrate an increasing incidence in several countries. Although clinical isolates of Francisella strains are sensitive to currently used antibiotics, engineered or horizontal acquisition of antibiotic resistance is a constant threat to public health. Therefore, the identification of antibiotics that target previously undrugged pathways is required to safeguard human health. An environmental pesticide that is registered for use in multiple countries, tolfenpyrad, shows promising activity to block Francisella growth; however, it is not a suitable antimicrobial candidate for use in vivo due to potential toxicity in humans and other animals. In this study, we applied a structure-activity relationship approach to tolfenpyrad to generate compounds with improved antibacterial activity and reduced toxicity. Through screening of a library of derivatives, we identified analogs with improved therapeutic windows compared with tolfenpyrad. Although structural diversity exists among these analogs, they inhibit the growth of Francisella species but not other Gram-negative or Gram-positive species. These compounds block intramacrophage growth of F. novicida and pathogenesis in an in vivo arthropod model of infection. Although the biochemical activity of these drugs is unknown, they appear to target the same pathway as the parent molecule because F. novicida mutants that are resistant to tolfenpyrad are also resistant to its analogs. Taken together, these findings suggest that these tolfenpyrad-derived compounds comprise a new class of Francisella-targeted antimicrobials and merit further evaluation and development.

Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) - a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide.